processing carrot juice by selected nonthermal technologies

PROCESSING CARROT JUICE BY SELECTED NONTHERMAL TECHNOLOGIES

AS HURDLES

By

PRASHANT RAJ POKHREL

A dissertation submitted in partial fulfillment of

the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY

Department of Biological Systems Engineering

DECEMBER 2017

ii

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of PRASHANT

RAJ POKHREL find it satisfactory and recommend that it be accepted.

Gustavo V. Barbosa-Cánovas, Ph.D., Chair

Juming Tang, Ph.D.

Shyam S. Sablani, Ph.D.

iii

ACKNOWLEDGMENT

Firstly, I would like to express my deepest gratitude to my advisor Dr. Gustavo V. Barbosa-

Cánovas who has always supported and encouraged me to grow professionally in the field of food

engineering. I must say I am standing here today because of his continuous support, help, advice,

and encouragement. I extent my sincere gratitude to my doctorate committee members, Dr. Juming

Tang and Dr. Shyam S. Sablani for their advice and support in this research and giving me a chance

to grow professionally in this field in a vivid way.

I would also like to give a huge thanks to USDA National Needs Fellowship Program for

providing funding for my Ph.D. studies. I would like to acknowledge Mr. Frank Younce, the pilot

plant manager, for providing me training and other technical assistance. My warm appreciation to

my mentors: Dr. Daniela Bermúdez-Aguirre, Dr. Ilce Gabriela Medina-Meza, and Dr. Kanishka

Bhunia. Great thank goes to the interns Camille Boulet and Camilla Garcia-Jange as well as

visiting scholar Taíse Toniazzo for helping me in many studies. I also would like to thank my

friends and members of Food Engineering Club at WSU for the warm friendship during my stay

at Pullman, WA. I would like to thank the staffs from Biological Systems Engineering department

at WSU specially Joanna Dreger, Dorota Wilk, and Jonathan Lomber.

My deep respect and gratitude to my parents Mr. Jiv Narayan and Mrs. Kamala Pokhrel,

my brothers Ravi and Sudarshan Pokhrel and my sisters Sharada and Bandana Pokhrel for their

continuous encouragement and inspiration. I am grateful to my loving family members and in-

laws for their support during my doctorate studies. Finally, I would like to give a deepest thanks

to my better half, Sita Acharya Pokhrel, for her unwavering love and support.

iv

PROCESSING CARROT JUICE BY SELECTED NONTHERMAL TECHNOLOGIES

AS HURDLES

Abstract

by Prashant Raj Pokhrel, Ph.D.

Washington State University

December 2017

Chair: Gustavo V. Barbosa-Cánovas

Nonthermal food processing technologies such as High Pressure Processing (HPP), Pulsed Electric

Fields (PEF), and Ultrasound (US) are novel juice pasteurization methods. The objective of this

study was to use these technologies in combination with other microbial stress factors (Mild heat,

Nisin, and pH) in the mild-pasteurization of carrot juice. Microbiological inactivation and quality

attributes retention were studied for selected combinations. Various processes were identified that

rendered equivalent microbial load reduction and those processes were compared for energy

consumption and quality retention.

The ultrasound (37.87 W/cm2) processing of carrot juice at 58°C, was found to be effective

in E. coli inactivation without impacting quality attributes. Mathematical modeling of the

inactivation’s curve shows that Weibull and a biphasic model to be good fits to predict survivors.

Further studies on ultrasound in combination with nisin (25 and 50 ppm) and mild temperatures

(35 and 50°C) show synergism between these three factors.

High pressure was combined with moderate heat and nisin to explore the inactivation of

gram-positive and negative bacteria. The addition of nisin at 25 and 50 ppm at 20°C did not impact

v

the lethal effect at mild-pressures (≤300 MPa). However, for the same level of pressures, a

synergistic effect between nisin and pressure was found at 35°C. The microbial inactivation by

combining HPP and nisin was further enhanced by increasing the temperature to 50°C. High

pressure treatment was also carried out for carrot juice mixed with orange juice in different

proportion. The developed interacting relations between pressure, pH, and processing time on

microbial inactivation could be useful in predicting the inactivation at different pHs.

The PEF processing of carrot-orange juice blend shows the application of High Electric

Field-Low Frequency was found to be more efficient than Low Electric Field-High Frequency in

microbial inactivation. The application of mild temperatures (35 and 50°C) and nisin (25 and 50

ppm) enhanced the inactivation by both processes without significantly impacting quality

attributes.

Findings from the present study will be useful in designing mild pasteurization processes

for fruit and vegetable juices using nonthermal technologies such as HPP, PEF, and Ultrasound

together with other selected hurdles.

vi

TABLE OF CONTENTS

Page

ACKNOWLEDGMENT................................................................................................................ iii

ABSTRACT ................................................................................................................................... iv

LIST OF TABLES ........................................................................................................................ xv

LIST OF FIGURES ................................................................................................................... xviii

INTRODUCTION .......................................................................................................................... 1

1. Background and Problem Statement ....................................................................................... 1

2. Hypothesis and Objectives ...................................................................................................... 3

3. Dissertation outline .................................................................................................................. 4

References ....................................................................................................................................... 5

CHAPTER ONE ............................................................................................................................. 7

A REVIEW ON NONTHERMAL PROCESSING OF FRUIT AND VEGETABLE JUICES ..... 7

1. Introduction ............................................................................................................................. 7

2. Microbial outbreaks in juices .................................................................................................. 8

3. Commercial juice processing................................................................................................... 9

4. Novel approaches for juice processing .................................................................................. 10

4.1 High pressure processing ............................................................................................... 10

4.1.1 HPP principles of operation .................................................................................... 11

4.1.2 Microbiological inactivation by HPP...................................................................... 11

4.1.3 Effect of HPP on physicochemical characteristics of juices ................................... 16

4.2 Pulsed electric fields....................................................................................................... 18

vii

4.2.1 How does PEF work?.............................................................................................. 19

4.2.2 Microbiological inactivation by PEF ...................................................................... 20

4.2.3 Effect of PEF on physicochemical characteristics of juices ................................... 23

4.3 Power ultrasound ............................................................................................................ 26

4.3.1 Power ultrasound and how it works ........................................................................ 26

4.3.2 Microbiological inactivation by ultrasound ............................................................ 27

4.3.3 Effect of ultrasound on physicochemical characteristics of juices ......................... 30

4.4 Combination of processes .............................................................................................. 33

4.4.1 Ultrasound and high pressure processing ............................................................... 33

4.4.2 Ultrasound and pulsed electric fields ...................................................................... 35

5. Final remarks ......................................................................................................................... 36

References ..................................................................................................................................... 38

CHAPTER TWO .......................................................................................................................... 54

ENGINEERING ASPECTS OF NOVEL TECHNOLOGIES FOR BEVERAGE PROCESSING

....................................................................................................................................................... 54

1. Introduction ........................................................................................................................... 54

2. High pressure processing ....................................................................................................... 55

2.1 HPP Equipment .............................................................................................................. 55

2.2 Compression heating and its effect ................................................................................ 58

2.3 Energy consumptions ..................................................................................................... 60

2.4 Packaging requirements ................................................................................................. 62

2.5 Factors affecting the process .......................................................................................... 63

2.6 Advantages and limitations of HPP ................................................................................ 68

viii

3. Pulsed electric fields .............................................................................................................. 69

3.1 PEF equipment ............................................................................................................... 70

3.2 Electric fields generation ................................................................................................ 74

3.3 Energy consumption ....................................................................................................... 75


3.5 Advantages and limitations of PEF ................................................................................ 80

4. Ultrasound ............................................................................................................................. 81

4.1 Ultrasound equipment .................................................................................................... 83

4.2 Propagation and attenuation of ultrasonic waves ........................................................... 85

4.3 Energy consumption ....................................................................................................... 86


4.5 Advantages and limitations of Ultrasound ..................................................................... 91

5. Final Remarks ........................................................................................................................... 92

References ..................................................................................................................................... 93

CHAPTER THREE .................................................................................................................... 101

COMBINED EFFECT OF ULTRASOUND AND MILD TEMPERATURES ON THE

INACTIVATION OF E. coli IN FRESH CARROT JUICE AND CHANGES ......................... 101

ON ITS PHYSICOCHEMICAL CHARACTERISTICS ........................................................... 101

Abstract ....................................................................................................................................... 101

1. Introduction ......................................................................................................................... 102

2. Materials and methods ......................................................................................................... 104

2.1 Preparation of carrot juice ................................................................................................. 104

2.2 Culture preparation ............................................................................................................ 105

ix

2.3 Ultrasound treatment ......................................................................................................... 105

2.4 Microbiological analysis ................................................................................................... 106

2.5 Microbial inactivation kinetics ..................................................................................... 107

2.5.1 First-order ................................................................................................................... 107

2.5.2 Weibull model ............................................................................................................ 107

2.5.3 Biphasic model ........................................................................................................... 108

2.6 Determination of color parameters .................................................................................... 108

2.7 Determination of pH, soluble solids content, and acidity ................................................. 109

2.8 Determination of phenolic compounds, ascorbic acid, and total carotenoid .................... 109

2.9 Statistical analysis ............................................................................................................. 110

3. Results and discussion ............................................................................................................ 111

3.1 E. coli Inactivation ............................................................................................................ 111

3.2 Mathematical modeling ..................................................................................................... 114

3.3 Effects of treatment on pH, soluble solids content, and acidity ........................................ 117

3.4 Effects of treatment on color parameters .......................................................................... 119

3.5 Effects of treatment on total carotenoid ............................................................................ 120

3.6 Effects of treatment on phenolic compounds .................................................................... 121

3.7 Effects of treatment on ascorbic acid ................................................................................ 122

4. Conclusions ......................................................................................................................... 123

References ................................................................................................................................... 124

CHAPTER FOUR ....................................................................................................................... 132

ON THE INACTIVATION OF Listeria innocua IN CARROT JUICE BY COMBINING

ULTRASOUND, HEAT, AND NISIN ...................................................................................... 132

x

Abstract ....................................................................................................................................... 132

1. Introduction ......................................................................................................................... 133

2. Materials and Methods ........................................................................................................ 135

2.2 Culture Preparation ........................................................................................................... 135

2.3 Ultrasound Treatment ........................................................................................................ 135

2.4 Survivor Analysis .............................................................................................................. 136

2.6 Optimum Processing Conditions and Quality Evaluation................................................. 138

3.1 Effect of Ultrasound on Listeria Inactivation ................................................................... 139

3.2 Combined Effect of Ultrasound and Temperature on Listeria Inactivation...................... 141

3.3 Combined Effect of Ultrasound, Temperature, and Nisin on Listeria Inactivation .......... 143

3.4 Energy Consumption during Processing ........................................................................... 146

3.5 Selection of Optimum Processing Conditions ...................................................................... 148

3.6 Effect of Processes on Total Aerobic Mesophiles ................................................................ 149

3.7 Evaluation of Effects on Quality Attributes.......................................................................... 150

3.7.1 pH, TSS, and Total Carotenoid Content ............................................................................ 150

3.7.2 Color Parameters ................................................................................................................ 152

4. Conclusions ......................................................................................................................... 153

References ................................................................................................................................... 155

CHAPTER FIVE ........................................................................................................................ 159

INACTIVATION OF Listeria innocua AND Escherichia coli IN CARROT JUICE BY

COMBINING HIGH PRESSURE PROCESSING, NISIN, AND MILD .................................. 159

THERMAL TREATMENTS ...................................................................................................... 159

1. Introduction ......................................................................................................................... 160

xi


2.1 Culture preparation ........................................................................................................ 162

2.2 Sample preparation ........................................................................................................ 162

2.3 High Pressure Processing (HPP) ................................................................................... 164

2.4 Enumeration of viable E. coli and L. innocua ............................................................... 165

2.5 Quality evaluation and storage studies .......................................................................... 165

2.6 Energy calculation ......................................................................................................... 168

2.7 Statistical analysis.......................................................................................................... 168

3. Results and Discussion ........................................................................................................ 169

3.1 Effect of process on the inactivation of L. innocua and E. coli ..................................... 169

3.2 Effect of treatments on the quality of fresh carrot juice ................................................ 175

3.3 Effect on pH, total soluble solids (TSS), and turbidity ................................................. 176

3.4 Effect on color characteristics ....................................................................................... 177

3.5 Effect on bioactive compounds ..................................................................................... 178

3.6 Effect of process on energy consumption ...................................................................... 182

3.7 Storage studies ............................................................................................................... 182

4. Conclusions ............................................................................................................................. 189

References ................................................................................................................................... 191

CHAPTER SIX ........................................................................................................................... 197

INACTIVATION OF Listeria innocua IN CARROT-ORANGE JUICE BLENDS BY HIGH

HYDROSTATIC PRESSURE ................................................................................................... 197

Abstract ....................................................................................................................................... 197

1. Introduction ......................................................................................................................... 198

xii


2.1 Sample Preparation ........................................................................................................... 200

2.2 Culture Preparation and Inoculation ................................................................................. 201

2.3 High Pressure Processing .................................................................................................. 201

2.4 Analysis of Survivors ........................................................................................................ 202

2.5 Determination of Color and Soluble Solid Contents ......................................................... 202

2.6 Ascorbic Acid .................................................................................................................... 202

2.7 Total Soluble Phenolic ...................................................................................................... 203

2.8 Total Carotenoid ................................................................................................................ 203

2.9 Quantification of Energy requirements ............................................................................. 203

2.10 Storage study ................................................................................................................... 205

2.11 Data Analysis .................................................................................................................. 205

3. Results and Discussion ........................................................................................................ 205

3.1 Inactivation of L. innocua ................................................................................................. 205

3.2 Multivariate Analysis ........................................................................................................ 208

3.3 Energy consumption during processing ............................................................................ 209

3.4 Quality evaluation ............................................................................................................. 210

3.4.1 Color characteristics ................................................................................................... 210

3.4.2 Total carotenoid content ............................................................................................. 211

3.4.3 Ascorbic acid .............................................................................................................. 212

3.5 Quality changes during storage ......................................................................................... 214

3.5.1 Microbial growth during storage ................................................................................ 214

3.5.2 Changes in pH and TSS during storage ...................................................................... 216

xiii

3.5.3 Changes in color of juice during storage .................................................................... 219

Conclusions ................................................................................................................................. 223

References ................................................................................................................................... 225

CHAPTER SEVEN .................................................................................................................... 229

PROCESSING OF CARROT-ORANGE JUICE BLEND BY PULSED ELECTRIC FIELDS

AND NISIN AT VARIOUS TEMPERATURES ....................................................................... 229

Abstract ....................................................................................................................................... 229

1. Introduction ......................................................................................................................... 230


2.1 Sample preparation ............................................................................................................ 231

2.2 Culture preparation and inoculation .................................................................................. 232

2.3 PEF processing .................................................................................................................. 232

2.4 Analysis of survivors ......................................................................................................... 233


2.5.1 Total aerobic mesophiles ............................................................................................ 234

2.5.2 pH, TSS, and color ..................................................................................................... 234

2.5.3 Total carotenoid content ............................................................................................. 234

2.5.4 Ascorbic acid .............................................................................................................. 235

2.6 Energy calculations ........................................................................................................... 235

2.7 Data analysis ..................................................................................................................... 235

3. Results and discussion ......................................................................................................... 236

3.1 Rise in temperature during processing .............................................................................. 236

3.2 Combined effect of PEF and heat ..................................................................................... 237

xiv

3.3 Combined effects of PEF and nisin ................................................................................... 239

3.4 Combined effect of PEF, heat, and nisin ........................................................................... 240


3.5.1 Effect of processes on aerobic mesophiles ..................................................................... 242

3.5.2 Effect on pH, TSS, and Color ........................................................................................ 243

3.5.3 Effect on total carotenoid content and ascorbic acid...................................................... 246

4. Conclusions ......................................................................................................................... 246

References ................................................................................................................................... 248

FINAL REMARKS .................................................................................................................... 251

FUTURE STUDIES.................................................................................................................... 253

xv

LIST OF TABLES

Table 1. Microbial outbreaks in fruits and vegetables. ................................................................... 8

Table 2. Effect of HPP on Microbial Inactivation ........................................................................ 15

Table 3. Effect of HPP on physical and chemical characteristics of juices. ................................. 17

Table 4. Effect of PEF on microbial inactivation. ........................................................................ 22

Table 5. Effect of PEF on physicochemical characteristics of juice............................................. 24

Table 6. Microbial inactivation in juices by Ultrasound. .............................................................. 29

Table 7. Effect of ultrasound on juice physiochemical characteristics. ........................................ 32

Table 8. Adiabatic increments in temperature during HPP (de Heij et al., 2003). ....................... 65

Table 9. Statistical comparison of three different mathematical models: First-order, Weibull, and

Biphasic based on R-square, Root mean square error (RMSE), Bias factor (Bf), Accuracy factor

(Af), and F-test. ........................................................................................................................... 117

Table 10. Physicochemical parameters of carrot juice before and after processing ................... 118

Table 11. Final temperature of the juice after ultrasound processing for 5 min at different

amplitudes ................................................................................................................................... 136

Table 12. Effect of nisin concentration on the inactivation of L. innocua at room temperature

(20°C). ......................................................................................................................................... 144

Table 13. Acoustic energy density and ultrasonic intensity at different amplitude levels (Values

are the average of three replicates ± standard deviation). ........................................................... 147

Table 14. Processing conditions that results in at least a 5 log reduction of L. innocua ............ 148

xvi

Table 15. Change in pH, TSS, and carotenoid content of juice before and after processing under

different treatment conditions ..................................................................................................... 152

Table 16. Change in Lightness, Hue angle, Chroma, and ΔE of the juice before and after processing

with different treatment conditions ............................................................................................. 153

Table 17. Optimum processing conditions that led to at least 5 log reduction of L. innocua and E.

coli. Combination ‘a’ is control or fresh untreated juice. ........................................................... 176

Table 18. Effect of combination of HPP, temperature, and nisin on pH, soluble solids, and

turbidity. ...................................................................................................................................... 177

Table 19. Effect of combinations of HPP, temperature, and nisin on the color characteristics of

carrot juice .................................................................................................................................. 178

Table 20. Effect of selected combination of treatments on carotenoids, phenolic compounds and

ascorbic acid................................................................................................................................ 180

Table 21. Compression work and total energy consumption during processing ........................ 182

Table 22. Change in pH, Total soluble solids and Turbidity of carrot juice during storage at 4°C.

..................................................................................................................................................... 186

Table 25. Change in color characteristics of carrot juice during storage at 4°C. ....................... 188

Table 24. Main effect and interaction effect of each factor on L. innocua inactivation ............. 208

Table 25. Energy consumption in HPP at different pressure and processing time ..................... 209

Table 26. Color of juice before and after processing. ................................................................. 211

xvii

Table 27. Results obtained for the quantification of ascorbic acid, carotenoid and phenolic content.

..................................................................................................................................................... 213

Table 28. Processing parameters for PEF ................................................................................... 233

Table 29. Temperature rise during pulsed electric field processing of carrot-orange juice blends

..................................................................................................................................................... 236

Table 30. Combined effect of PEF, mild heat, and nisin on the inactivation of L. innocua in a

carrot-orange juice blend ............................................................................................................ 241

Table 31. Change in pH and TSS of juice before and after PEF processing .............................. 244

Table 32. Change in color characteristics of juice before and after PEF treatment .................... 245

xviii

LIST OF FIGURES

Figure 1. Structural and functional changes in microorganisms at different pressures (Source: Lado

and Yousef, 2002) ......................................................................................................................... 13

Figure 2. Electroporation of a microbial cell by PEF (Source: Vega-Mercado et al., 1996). ...... 21

Figure 3. Cavitation in ultrasound (a) Bubble formation; (b) Bubble growth; (c) Bubble collapse

(Source: Abdullah and Chin, 2014). ............................................................................................. 27

Figure 4. Combination of ultrasound and pressure (Source: Lee et al., 2009). ............................ 35

Figure 5. Operation of HPP (Source: http://www.hiperbaric.com/). ............................................ 57

Figure 6. Hiperbaric HPP system Hiperbaric-135 (Source: http://www.hiperbaric.com/). .......... 57

Figure 7. Avure’s AV-30 HPP unit (Source: http://www.avure-hpp-foods.com/). ...................... 58

Figure 8. Time–temperature and pressure profiles of water at an initial temperature of 24.2ºC and

set pressure of 300 MPa during HPP (Source: Patazca et al., 2007). ........................................... 65

Figure 9. Electroporation in microbial cell after PEF treatment. .................................................. 70

Figure 10. Schematic diagram of a pulsed electric fields system ................................................. 71

Figure 11. Pilot scale PEF systems: a. ELEA technologies (15 kW) and b. Diversified technologies

Inc. (DTI) (25 kW) (Source: http://elea-technology.de/ and http://www.divtecs.com/) .............. 71

Figure 12. Pulse forming network (PFN) in PEF system ............................................................. 72

Figure 13. PEF treatment chamber ............................................................................................... 74

Figure 14. Schematic diagram of ultrasound processing .............................................................. 81

xix

Figure 15. Creation of stable cavitation bubbles; and creation and the collapse of transient

cavitation bubbles. (a) displacement; (b) transient cavitation; (c) stable cavitation; (d) pressure

(Source: Santos et al., 2009). ........................................................................................................ 83

Figure 16. Ultrasound equipment: Lab scale (Left) and Industrial scale (Right) (Source:

www.hielscher.com) ..................................................................................................................... 84

Figure 17. Effect of frequency on the size of bubbles. (Source: http://www.ctgclean.com/). ...... 88

Figure 18. Inactivation kinetics of E. coli: (a) inactivation curve at different temperatures (50, 54,

and 58°C); Modeling the inactivation kinetics of E. coli by ultrasound at 50 (b), 54 (c), and 58°C

(d) using first-order, Weibull, and Biphasic model. ................................................................... 113

Figure 19. Carotenoid content of juice before and after processing, control indicates the fresh juice

without any treatment, US+50°C, US+54°C, US+58°C are three different ultrasound treatment at

50, 54 and 58°C respectively. There was non-significant (p>0.05) increment in total carotenoid

content after each treatment. ....................................................................................................... 121

Figure 20. Effect of ultrasound amplitude on inactivation of L. innocua in carrot juice at 20°C (left)

and 35°C (right). ......................................................................................................................... 140

Figure 21. Effect of temperature at different amplitude levels on the inactivation of L. innocua in

carrot juice (Data are mean of three replicates) .......................................................................... 143

Figure 22. Effect of different nisin concentration on inactivation of L. innocua at 35°C and 50°C

and at 80 and 100% amplitude level. Data are mean of three replicates .................................... 146

Figure 23. Temperature rise during processing at different amplitudes. Error bar represent the

standard deviation of three replicates ......................................................................................... 147

xx

Figure 24. Inactivation of mesophiles in carrot juice by selected combination of ultrasound

treatment. No significant difference (p>0.05) was found between selected equivalent processes on

the inactivation of mesophiles; Control indicates fresh juice without any treatments (a, b, c, d e as

shown in Table 14). Data are mean of three replicates ............................................................... 150

Figure 25. Process flow diagram for High Pressure Processing of nisin-incorporated carrot juice at

mild temperatures........................................................................................................................ 164

Figure 26. Effect of increased pressure at room temperature (20°C) on reduction of L. innocua and

E. coli. ......................................................................................................................................... 170

Figure 27. The combined effect of HPP, nisin, and heat on microbial inactivation. Data represent

the mean of three replicate .......................................................................................................... 173

Figure 28. Growth of aerobic mesophilic bacteria in carrot juice during storage at 4°C when

exposed to different treatments: b: 200 MPa, 50 ppm, 50°C, c: 300 MPa, 25 ppm, 35°C, d: 400

MPa, 0 ppm, 35°C, e: 500 MPa, 0 ppm, 20°C. ........................................................................... 184

Figure 29. Temperature and pressure profile during processing at 400 MPa for 1 min used to

determine δT/δt and δP/δt required for the energy calculation ................................................... 204

Figure 30. Inactivation of L. innocua in carrot-orange juice blends at 200 MPa, 300 MPa and 400

MPa over 5 minutes .................................................................................................................... 206

Figure 31. The growth of total aerobic mesophilic bacteria in the control (un-processed) and high

pressure treated juice blends during storage at 4°C .................................................................... 216

Figure 32. Change in pH and TSS of the juice blends (Control and Processed) during storage at

4°C for 28 days. .......................................................................................................................... 219

xxi

Figure 33. Change in color characteristic of the juice blends during storage at 4°C for 28 days.

..................................................................................................................................................... 223

Figure 34. Inactivation of L. innocua and naturally occurring mesophiles in carrot-orange juice

blends by pulsed electric fields. (ND: Not Detectable, below the detection limit) (PI: LEHF; PII:

HELF) ......................................................................................................................................... 239

Figure 35. Inactivation of naturally occurring mesophilic bacteria in carrot-orange juice by PEF

and mild heat. (PI: LEHF; PII: HELF) ....................................................................................... 243

Figure 36. Effect of selected process on carotenoid and ascorbic acid content of juice ............. 246

1

INTRODUCTION

Thermal pasteurization is the most commonly used technique for the treatment of fresh fruit and

vegetable juices. This process is successful in inactivating pathogenic and spoilage

microorganisms as well as enzymes. However, thermal processes can negatively affect the

nutritional quality and sensory attributes of juices (Wolbang et al., 2008). Low-acid and thermo-

sensitive juices such as carrot juice are affected by thermal processing; in carrot juice, it can lead

to losses of color, beta carotene, ascorbic acid, and phenolic compounds among other negative

effects (Patterson et al., 2012). Bioactive compounds present in this juice have high economic and

health significance. Many researchers have reported that thermal processing of fruits juices causes

major degradation of heat-sensitive bioactive compounds (Weemaes et al., 1998, Ferrari et al.,

2011). Thermal processing easily degrades anthocyanin content and antioxidant activity in juices.

Hager et al., (2008) observed that pasteurization of blackberry using thermal processing (90°C for

3 min) caused a 67% and 55% reduction in total monomeric anthocyanin content and antioxidant

activity respectively. To preserve the nutrient and sensory attributes of juice, there is a need for

new methods which can inactivate harmful microorganisms with minimum energy consumption

and without significantly affecting the quality of the juice. Novel nonthermal technologies such as

high pressure processing, pulsed electric fields and ultrasound in combination with temperature,

pH reduction, and antimicrobials addition can overcome the shortcomings of traditional processes.

1. Background and Problem Statement

Over the past few decades, several bacterial outbreaks have been associated with fruit and

vegetable juices. Most of these juice-related outbreaks have been associated with E. coli O157:H7,

Listeria monocytogenes, and Salmonella spp. (Jain et al., 2009; CDC 2015). Due to outbreaks

2

associated with juices, the United States Food and Drug Administration (USFDA) passed

legislation that requires all juice processors to follow a Hazard Analysis Critical Control Point

(HACCP) plan. According to the legislation, any commercial process should be able to achieve at

least 5 log reduction of most resistant pathogens associated with the particular juice (USFDA,

2001). Low acid juices (pH > 4.6), such as carrot juice, are considered risky and the FDA considers

such juices hazardous as they support the growth of pathogens of significant concern (Pilavtepe-

Çelik, 2013). As stated earlier, thermal processing can inactivate the pathogenic and spoilage

microorganisms efficiently but it has negative consequences such as loss of organoleptic quality

and loss of heat sensitive bioactive compounds.

Nonthermal food processing technologies such as high pressure processing (HPP), pulsed

electric fields (PEF) and ultrasound are novel methods to pasteurize fresh fruit and vegetable

juices. Among these three technologies, HPP has been already used at industrial setting to process

juice and other beverages but the level of pressure required and, in some cases, long treatment

times (>5 min) makes the process less efficient. Regarding PEF, this technology has been used at

industrial level for juice processing as well as for extraction of bioactive compounds and to create

pores in tissues in order to facilitate other processes such as dehydration. Microbial inactivation in

juice by PEF can be further enhanced by the incorporation of other stress factors making the overall

process quite efficient. Similarly, ultrasound technology has also been used at commercial level

for extraction of intracellular components. Ultrasound has the potential for the inactivation of

microorganisms due to its cavitation effect on the membrane cells, but the level of inactivation that

it renders is below the target level when it is used alone. There are many evidences ultrasound and

heat have strong synergism in microbial inactivation. All the above mentioned nonthermal

technologies could be combined with natural antimicrobial and thermal energy to increase the

3

efficacy of microbial inactivation and reduce energy consumption making those processes cost

effective.

2. Hypothesis and Objectives

By using selected nonthermal processing technologies in combination with other microbial stress

factors such as thermal energy, pH reduction, and nisin addition the required level of microbial

inactivation could be achieved in shorter time and with less energy input than with conventional

thermal treatments. A reduction in the energy input/consumption and processing time could

contribute to high retention of food quality attributes.

The main objectives of this research are:

Inactivate microorganisms of concerns in carrot juice by selected nonthermal processing

technologies using a multi hurdle approach

Study changes in physicochemical characteristics and retention of bioactive compounds in

carrot juice after selected individual and combined processes

Identify equivalent processes in terms of microbial inactivation to process carrot juice to

compare their effect on nutrient retention and energy consumption

Study changes in microbiological and physicochemical characteristics of processed juice

blends during storage

Reduce the energy consumption and processing time in selected nonthermal technologies

in the processing of carrot juice and carrot-orange blends

Find optimum processing conditions for mild-pasteurization of carrot juice for the studied

nonthermal technologies and their combinations with other hurdles

4

3. Dissertation outline

This dissertation consists of eight chapters:

Chapter I is a review on the safety and quality aspects of fruit and vegetable juices while

processed by high hydrostatic pressure, pulsed electric fields and ultrasound together with some

microbiological stress factors.

Chapter II is another literature review, but this time on the engineering aspects of beverage

processing by novel technologies. Main topics covered in this study include equipment design for

each technology, energy consumption during processing, factors affecting the efficacy of each

process and advantages/limitation of the processes.

Chapter III presents the combined effect of ultrasound and mild temperature on the inactivation

of E. coli in carrot juice and changes on its physicochemical characteristics.

Chapter IV deals with the physicochemical and microbiological quality changes in thermo-

sonicated carrot juice during storage at 4°C.

Chapter V explores the inactivation of Listeria innocua in carrot juice by combining ultrasound,

heat, and nisin.

Chapter VI analyses the effect of combining high pressure processing, nisin, and mild temperature

on the inactivation of Listeria innocua and Escherichia coli.

Chapter VII deals with the inactivation of Listeria innocua in carrot-orange juice blends by high

hydrostatic pressure.

Chapter VIII studies the effect of pulsed electric fields processing on the inactivation of Listeria

innocua in a carrot-orange juice blend.

Final remarks and recommendations for future work are included at the end of this dissertation.

5

References

Patterson, M. F., McKay, A. M., Connolly, M., & Linton, M. (2012). The effect of high hydrostatic

pressure on the microbiological quality and safety of carrot juice during refrigerated

storage. Food Microbiology, 30(1), 205-212.

Wolbang, C. M., Fitos, J. L., & Treeby, M. T. (2008). The effect of high pressure processing on

nutritional value and quality attributes of Cucumis melo L. Innovative Food Science and

Emerging Technologies, 9(2), 196-200.

Pilavtepe‐Çelik, M. (2013). High hydrostatic pressure (HHP) inactivation of foodborne pathogens

in low‐acid juices. International Journal of Food Science and Technology, 48(4), 673-677.

Jain, S., Bidol, S. A., Austin, J. L., Berl, E., Elson, F., Williams, M. L., ... & Yu, P. A. (2009).

Multistate outbreak of Salmonella Typhimurium and Saintpaul infections associated with

unpasteurized orange juice—United States, 2005. Clinical Infectious Diseases, 48(8),

1065-1071.

CDC. (2015). Foodborne Outbreak Online Database (FOOD).

http://wwwn.cdc.gov/foodborneoutbreaks/Default.aspx. Accessed February 4, 2016

Weemaes, C. A., Ludikhuyze, L. R., Van den Broeck, I., Hendrickx, M. E., & Tobback, P. P.

(1998). Activity, electrophoretic characteristics and heat inactivation of

polyphenoloxidases from apples, avocados, grapes, pears and plums. LWT-Food Science

and Technology, 31(1), 44-49.

6

Hager, T. J., Howard, L. R., & Prior, R. L. (2008). Processing and storage effects on monomeric

anthocyanins, percent polymeric color, and antioxidant capacity of processed blackberry

products. Journal of Agricultural and Food Chemistry, 56(3), 689-695.

Ferrari, G., Maresca, P., & Ciccarone, R. (2011). The effects of high hydrostatic pressure on the

polyphenols and anthocyanins in red fruit products. Procedia Food Science, 1, 847-853.

7

CHAPTER ONE

A REVIEW ON NONTHERMAL PROCESSING OF FRUIT AND VEGETABLE

JUICES

1. Introduction

In conventional thermal processing of fresh fruit and vegetable juices, high temperature (60-90°C)

is used and the process is called or high-temperature short-time (HTST) processing ultra-high

temperature (UHT) pasteurization. This process is successful in preserving juice by inactivating

pathogenic and spoilage microorganisms, but at the same time, it degrades nutrient and sensory

attributes due to high temperature and/or long processing time. Recently, consumers are showing

more interest in preservative-free juice which has more fresh-like attributes. To preserve the

nutrient and sensory attributes of juice, there is a need for new methods which will inactivate

pathogenic microorganisms, but not affect the quality of processed juice. The use of nonthermal

processing technologies such as ultrasound (US), high pressure processing (HPP), and pulsed

electric fields (PEF), in combination with moderate temperatures and/or antimicrobial agents, can

overcome shortcomings of conventional thermal processes. The above mentioned nonthermal

processing technologies have the capacity to inactivate food-borne pathogens, and in most cases

to preserve physicochemical and organoleptic properties of foods (Knorr et al., 2011a; Rawson et

al., 2011). Due to the synergistic or additive effects of these technologies, together with hurdles

such as the addition of antimicrobial agents or reduction of pH, the desired level of microbial

inactivation can be achieved in a very short time with or without application of moderate heat

(Nguyen and Mittal, 2007; McNamee et al., 2010; Chung and Yousef, 2010).

8

This chapter reviews the effects of HPP, PEF, and US, in combination with various hurdles,

on microbial inactivation in fresh juices. It also provides an overview of recent findings on how

this technology affects the quality of fresh juice including color, ascorbic acid, phenolic

compounds, carotenoids, antioxidant activity, vitamins, and other attributes.

2. Microbial outbreaks in juices

Over the past few decades, several bacterial outbreaks have been linked with fruit and vegetable

products (Table 1). Most of these juice-related outbreaks have been associated with, Listeria

monocytogenes and E. coli O157:H7 (Jain et al., 2009; CDC 2015). Due to various outbreaks with

juices, the United States Food and Drug Administration (USFDA) passed legislation that requires

all juice processors to follow a Hazard Analysis Critical Control Point (HACCP) plan. According

to the legislation, any commercial process should be able to achieve at least 5 log reductions of

most resistant pathogens in particular juice (USFDA, 2001). Low acid juices (pH > 4.6), such as

carrot juice, are considered risky, and the FDA considers such juices hazardous, as they support

the growth of pathogens (Pilavtepe-Çelik, 2013).

Table 1. Microbial outbreaks in fruits and vegetables (Source: CDC, 2015).

Year Pathogen Media Number of cases Area of outbreak

1993 Clostridium botulinum Carrot juice 1 Washington, USA

1993

Escherichia coli

(ETEC) Carrots 121 New Hampshire, USA

2000 Escherichia coli Watermelon >41 Milwaukee, USA

9

2006

Escherichia coli

O157:H7 Fresh Spinach 199 with 3 deaths 26 states of USA

2006

Salmonella

Typhimurium Tomato 183 21 states of USA

2011 Listeria monocytogenes Cantaloupe

146 with 30

deaths 28 states of USA

2011

Escherichia coli

O157:H7

Romaine

lettuce 56 9 states of USA

2012

Escherichia coli

O157:H7

Spinach and

Spring Mix

Blend

33 5 states of USA

2012 Salmonella Braenderup Mangoes 127 15 states of USA

2013 Salmonella Saintpaul Cucumbers 84 18 states of USA

2014 Salmonella Newport Cucumbers 275

29 states and District

of Columbia, USA

2015 Salmonella Poona Cucumbers 907 with 6 deaths 40 states of USA

3. Commercial juice processing

Thermal pasteurization is the most commonly used technique for the treatment of fresh fruit and

vegetable juices and this process is successful in inactivating pathogenic and spoilage

microorganisms. However, thermal processing can adversely impact the nutritional quality and

sensory attributes of juice (Wolbang et al., 2008). Low-acid and thermo-sensitive juices such as

10

carrot juice are affected by thermal processing; in carrot juice, it can lead to losses of color, beta

carotene, ascorbic acid, and phenolic compounds, among other negative effects (Patterson et al.,

2012). The bioactive compounds present in the juice have high economic and health significance.

Many researchers have reported that thermal processing of fruits juices causes major degradation

of heat-sensitive bioactive compounds (Weemaes et al., 1998, Ferrari et al., 2011). Thermal

processing can rapidly degrades heat sensitive anthocyanin content and antioxidant activity in juice

as well as other products (Hager et al., 2008). In order to retain those heat sensitive bioactive

compounds, novel technologies for juice processing has been proposed.

4. Novel approaches for juice processing

Due to the significant negative consequences of thermal processing, there was a need for novel

juice processing methods. High pressure processing, pulsed electric fields, and ultrasound are some

of the novel processing methods that have shown promising results in juice processing. These three

technologies are reviewed in this section.

4.1 High pressure processing

High pressure processing (HPP) is a mature nonthermal technology with origins in the chemical

processing industry. The FDA has approved this technology as a viable pasteurization method, and

it can be applied for preservation purposes as well as for extraction of essential bioactive

compounds from food. HPP has shown success in microbial and enzymatic inactivation at elevated

pressures up to 900 MPa. Liquid and solid foods, after packaging, are pressurized to between 50

and 900 MPa. In most cases, HPP inactivates spoilage and pathogenic microorganisms with

minimal changes in sensory attributes and nutrient loss compared to the same foods prepared with

11

conventional thermal processing (Timmermans et al., 2011). Juices, smoothies, guacamole,

sauces, ham, and seafood are some of the commercially available products processed by high

pressure.

4.1.1 HPP principles of operation

HPP can be largely described by the isostatic principle and Le Châtelier’s principle. The isostatic

principle explains the manner in which HPP transmits pressure uniformly and instantaneously

through all contents of the treatment chamber, regardless of whether the product being treated is

contained within packaging or is directly in contact with the pressure medium. Le Châtelier’s

principle states that a process that decreases the volume upon which it acts will be accelerated by

compression, i.e. application of high pressure. Combined, these principles explain why the

processing time of HPP does not depend on the volume, size, or packaging (or lack thereof) of the

product being treated, or on its location within the treatment chamber.

4.1.2 Microbiological inactivation by HPP

Several studies on the inactivation of pathogenic and spoilage microorganisms using high pressure

have been published in recent years and have demonstrated that HPP is a preservation method that

effectively inactivates microorganisms in fruit and vegetable juices (Table 2). Processing pressure,

pressure holding time, decompression time, and treatment temperature have important roles in the

treatment of fruit products by HPP.

It is widely agreed that HPP disrupts non-covalent bonds within cell membranes and leads

to their permeabilization (Winter & Jeworrek, 2009; Knorr et al., 2011b). The pressure sensitivity

of microorganisms depends upon the species, strain and type of microorganism as well as their

12

growth phase; those in the stationary and lag phases are typically more resistant to HPP than those

in the exponential growth phase.

Both linear and nonlinear inactivation kinetics of microorganisms can be observed in HPP.

In the majority of microorganisms, first-order kinetics have been observed, whereas two-phase

inactivation is also common: the first phase affects treatment-sensitive and the second phase

treatment-resistant populations (Lee et al., 2001). Exposure to high pressure causes filament

formation in microbial cells and rupture of cell membranes; the growth and reproduction of

microbial cells are inhibited at high pressure.

The inactivation of microbial cells depends on several parameters including stage of

growth, type of microorganisms, processing time, and processing temperature. Due to the presence

of a peptidoglycan layer within their cell membrane, gram-positive bacteria have higher resistant

to pressure than gram-negative bacteria. Vegetative cells have been observed to be less sensitive

to HPP while in the stationary phase than while in the growth or exponential phases (Alpas et al.,

1999). Yeasts and molds are quite resilient, but can be inactivated by HPP using pressures on the

order of 200–600 MPa. Pressure damage to the cell or cytoplasmic membrane is the main cause of

HPP-related microbial inactivation (Yuste et al., 2001). HPP mainly causes alterations in cell

membrane permeability, and crystallization of membrane phospholipids. Application of high

pressure also has inhibitory effects on microbial enzymes which alter biochemical reactions

leading to protein denaturation, decrease of intracellular pH, and ultimately, inactivation.

High pressure processing is effective at room temperature as well as at elevated

temperatures. At mild temperatures, HPP has a minimal effect on sensory qualities associated with

texture, color, flavor, and nutrient retention. During high pressure processing, there is an increase

13

in temperature through compression heating. This increase in temperature during processing, in

addition to applied heat, can eliminate spore-forming bacteria (Margosch, 2005). Addition of heat

during high pressure processing is referred as Pressure Assisted Thermal Processing.

Figure 1. Structural and functional changes in microorganisms at different pressures (Source: Lado and

Yousef, 2002)

HPP can also be combined with antimicrobials as one of the hurdles typically used in food

preservation. Antimicrobials can be incorporated in the food matrix before processing, some

antimicrobials commonly used in liquid food products include nisin, lysozymes, and Lactobacillus

casei. Use of antimicrobials may reduce processing time, which in turn reduces energy

consumption.

14

The combined effect of HPP and Lactobacillus casei cell extract (as antimicrobial) on the

inactivation of Listeria monocytogenes in a food model has been explored by Chung and Yousef

(2010). This study combined HPP (350 MPa, 1–20 min) and Lactobacillus casei cell extract (32

AU/mL); this combination decreased the load of L. monocytogenes by >5 log CFU/mL, which

they described as a synergistic effect. A study by Sokołowska et al. (2012) on the processing of

apple juice using HPP and a peptide hurdle showed that a combination of HPP (200 MPa, 45 min)

and nisin (250 IU/mL) was capable of complete inactivation of Alicyclobacillus acidoterrestris

spores (>6 log reduction). Similarly, Zhao et al. (2013) used a combination of HPP and nisin to

achieve complete inactivation of natural yeast and mold in cucumber juice, applying a pressure of

500 MPa with 100 IU/mL nisin over 2 min, a clear synergistic effect. Table 2 presents examples

of microbial inactivation in different media by HPP.

Syed et al. (2013) found that slower decompression periods and faster compression periods

were more efficient at inactivating Escherichia coli O157:H7 in orange juice. Syed et al. (2012),

in contrast, concluded that slower compression and decompression at elevated temperatures (60

and 70ºC) were more efficient to inactivate Bacillus subtilis in the same medium. Pressure

treatment (200–600 MPa, 5–10 min) was capable of yielding a 2.5 log reduction in E. coli O157:H7

in pineapple juice (Buzrul et al., 2008), and HPP treatment of apple and orange juices at elevated

temperature (57 and 60°C, respectively) caused a 6-log reduction (Muñoz et al., 2007). This shows

that incorporation of mild temperature in HPP significantly increase the inactivation of

microorganisms.

15

Table 2. Effect of HPP on Microbial Inactivation

Microorganisms Media HPP conditions

Log

Reduction

References

L. Monocytogenes

Carrot

juice

500 MPa, 5.5 min,

20ºC

6 Patterson et al., 2012

E. coli

(ATCC 11775)

Apple

juice

600 MPa, 7 min,

21ºC

7 Moody et al., 2014

E. coli (ATCC

29055)

Apple

juice

400 MPa, 5 min,

25ºC

8 Ramasawamy et al., 2003

E. coli O157:H7

Manjo

juice

400 MPa, 10 min 6

Hiremath and

Ramaswamy, 2012

E. coli

(ATCC 11775)

Kiwifruit

juice

350 MPa, 5 min 5 Buzurul et al., 2008

B. licheniformis

Carrot

juice

600 MPa for 3 min

at 60°C

4.9

Tola and Ramaswamy,

2014

A. acidoterrestris

Orange

juice

200 MPa, 10 min,

at 65°C

2 Silva et al., 2012

B. coagulans

Tomato

pulp

600 MPa for 15

min at 60°C

5.7 Zimmermann et al., 2013

Byssochlamys

nivea

Pineapple

juice

550 MPa for 15

min at 40°C

3.9

da Rocha Ferreira et al.,

2009

16

Talaromyces

avellaneus

Apple

juice

600 MPa for 5 min

at 17°C

6 Voldřich et al., 2004

4.1.3 Effect of HPP on physicochemical characteristics of juices

Fruit and vegetable juices are rich sources of bioactive compounds such as ascorbic acid, phenolic,

and anthocyanin. These compounds have numerous health benefits hence, during processing, food

industry tries to preserve them (Kaşikçi and Bağdatlioğlu, 2016). The treatment of such juices by

HPP is reported to result in minimal changes in quality.

Varela Santos et al. (2012) found retention of anthocyanin, phenolic compounds, and color

of pomegranate juice after the application of 350 and 550 MPa at room temperature. The

anthocyanin content of blueberry juice has been shown to increase following HPP (600 MPa,

42°C) (Barba et al., 2013). Another study on high-pressure treatment of pomegranate juice

concluded that HPP did not significantly affect its free anthocyanin content, whereas thermal

processing (85°C, 10 min) caused significant reductions (Alpas, 2013).

Phenolic compounds are another important class of bioactive compounds found in fruit and

vegetable juices. Many studies show retention of phenolic compounds after high pressure

treatment. Ferrari et al. (2010) found a 41% increase in the polyphenol content of pomegranate

juice after high pressure treatment at 400 MPa, 10 min, 50°C while thermal processing alone

(50ºC/10 min) showed no effect. This indicates that HPP not only retains base levels of phenolic

compounds, but also helps extract them from the tissue, thereby increasing the available total

phenols in juice Another study on pomegranate juice found significant increases in its total

17

phenolic content after the application of 350 and 500 MPa for 30 to 150 seconds (Andrés et al.

2015).

The release of phenol from the tissue depends on the level of pressure applied. A study

conducted on HPP of strawberry puree showed that there were no changes in total phenolic content

by the application of 400-500 MPa, 15 min at 10-30°C, but when pressure was raised to 600 MPa,

keeping other factors constant, there was a significant increase in phenolic content (Patras et al.,

2009). Xi et al. (2009) found phenol extraction was independent of treatment time, depending only

on pressure; application of 200, 300, and 400 MPa resulted in 23, 26, and 30% increases in

extraction yield while changing the treatment time from 1 to 10 min had no significant effect.

Hence it can be said that HPP cause little or no degradation in bioactive compounds of juices at

room and slightly above temperature. Table 3 presents few studies on the retention of bioactive

compounds by HPP.

Table 3. Effect of HPP on physical and chemical characteristics of juices.

Bioactive

compounds

Media HPP conditions

Retention

(%)

References

Total phenolic

compounds

Mango

nectar

600 MPa, 1 min, 25°C 100 Liu et al., 2014

Total phenolic

compounds

Pomegranate

juice

400 MPa, 5 min, 20°C 103 Chen et al., 2013

Ascorbic acid Kiwi puree 500 MPa, 3 min, 20°C 94

Fernández-Sestelo et al.,

2013

18

Ascorbic acid

Mango

nectar

600 MPa, 1 min, 25°C 100 Liu et al., 2014

Ascorbic acid Longan juice 300 MPa, 30 min 90

Chaikham and

Apichartsrangkoon 2012

Total

carotenoids

Orange juice 400 MPa, 1 min, 40°C >100

Sánchez -Moreno et al.,

2005

L-ascorbic acid Orange juice 400 MPa, 1 min, 40°C 92.11

Sánchez-Moreno et al.,

2005

Total

flavanones

Orange juice 400 MPa, 1 min, 40°C >100

Sánchez-Moreno et al.,

2005

Ascorbic acid

Blueberry

juice

600 MPa, 5 min 91.82 Barba et al., 2013

Total phenolics

Blueberry

juice

600 MPa, 5 min >100 Barba et al., 2013

Anthocyanin

(C3G)

Mulberry

juice

200 MPa, 1 pass

homogenization, 4°C

61.2 Yu et al., 2014

4.2 Pulsed electric fields

Pasteurization of fruit and vegetable juices with pulsed electric fields (PEF) is another promising

yet challenging approach. Pulsed electric fields processing is gaining popularity as a food

processing treatment, presenting a nonthermal alternative to conventional pasteurization

processes. PEF has been investigated for liquid food decontamination for nearly 20 years, with

great success for juices, sauces, dairy products, and even liquid eggs.

19

A PEF processing system consists of several components: a control unit, a high voltage

pulse generator, and one or more treatment chambers. It can be operated in a continuous manner

and can be set up for high throughputs. As the processing time is short, there is a greater likelihood

of nutrient retention than in traditional thermal processing. This technology achieves inactivation

of microbial cells present in food by subjecting them to critical electric fields for a short period of

time.

With PEF technology, reductions of >5 log in common pathogens, including E. coli,

Listeria monocytogenes, and Salmonella spp. have been repeatedly demonstrated in a ranges of

food products, including those with particulates. Apple juice, orange juice and milk are some liquid

foods whose processing by PEF has been thoroughly explored (Zhang et al., 1995). Other

microorganisms of interest, such as Saccharomyces cerevisiae, Staphylococcus aureus,

Lactobacillus spp, Bacillus subtilis, Zygosaccharomyces bailii, etc., have been successfully

inactivated by this technology. PEF could be a substitute for conventional thermal processing for

the pasteurization of liquid foods, offering advantages including low energy consumption and short

processing times (see; Table 4)

4.2.1 How does PEF work?

PEF processing involves generation of high-intensity electric fields (>10 kV/cm) of short duration

followed by rapid electron discharge. These discharges take place into pumpable food products,

which are passing through two electrodes; the discharge generates a voltage difference across the

microbial cell membrane promoting the formation of irreversible pores (electroporation). These

pores allow the passage of essential cell components associated with its metabolism (lysis) and

thus promote the irreversible inactivation of the microorganism. The electric pulses are also

20

responsible for modifying intracellular enzymes and detaching the cytoplasm from the membrane.

These two additional factors contribute significantly to microbial inactivation.

4.2.2 Microbiological inactivation by PEF

PEF causes microbial inactivation through pore formation (electroporation) within the cell

membrane. The electric field intensity, treatment duration, and treatment temperature all play roles

in determining whether the formation of these pores is reversible. Exposure to an electric field

generates free charges on the surface of the microbial cell membrane, causing opposite charges on

the inner and outer membranes to attract each other. This attraction compresses the membrane,

reducing its thickness (Shamsi 2008). Thus, PEF-treated cells have weak and thin membranes,

which allow small molecules to more easily pass through them, potentially resulting in cell

membrane rupture (Wu, 2014). When the electric field intensity is increased there will be a greater

accumulation of surface charges that results into irreversible breakdown of the cell membrane

(Zimmerman, 1986).

The inactivation of microorganisms can also be explained on the basis of the osmotic

imbalance theory. This theory entails that microbial cell are electroporated when they are exposed

to an electric field causing leakage of ions intracellular constituents. This cause the cell membrane

to be permeable to water which eventually led to rupture and death of the cell (Shamsi, 2008).

21

Figure 2. Electroporation of a microbial cell by PEF (Source: Vega-Mercado et al., 1996).

Many studies on juice pasteurization by PEF claim that 3-4 log reduction of vegetative

pathogens in fruits and vegetable juice can be achieved by PEF alone (Table 4). Incorporation of

some selected hurdles might result in additional inactivation.

In one study, PEF processing (35 kV/cm electric field, 3 μs pulse width, 45 Hz) of cactus

juice reduced the naturally occurring total colony and yeast/mold counts below detectable levels

(Moussa-Ayoub et al., 2017). Inactivation of natural flora can increase a product’s shelf life

relative to fresh/untreated products. Simpson et al. (1995) reported that PEF-treated (50 kV/cm,

10 pulses, 2 µs pulse width, 45°C) apple juice had an extra week of shelf-life compared to untreated

apple juice (28 vs. 21 days).

Previous studies show that the efficacy of the PEF processing can be enhanced by

incorporation of stress factors such as reduction of media pH, addition of antimicrobials, or an

increase in processing temperature. Studies have shown that the combination of PEF with a wide

range of antimicrobial hurdles, including antifungal peptides, bacteriocins, essential oils, organic

acids, and spices, can lead to improved microbial inactivation in fruit juices (Liang et al., 2002;

Nguyen and Mittal, 2007; Mosqueda-Melgar et al., 2008).

22

According to Liang et al. (2002) there was 5.9 log reduction of Salmonella typhimurium in

freshly squeezed orange juice (without pulp) when treated by PEF (90 kV/cm, 50 pulses, at 55ºC)

but when nisin (100 IU/mL of orange juice) or lysozyme (2,400 IU/mL) was added to the juice

before PEF treatment, there was additional 2.75 log cycles. Altunakar (2007) tested the use of a

variety of organic antimicrobials (e.g. cinnamic acid, hydrogen peroxide) as pretreatments before

applying PEF (40 kV/cm, 2.5 µs pulse width); these pretreatments were capable of increasing

treatment lethality by from 5 to up to 7 log cycles. In addition, Jin et al. (2014) found that

pomegranate juice processed by PEF (35kV/cm field strength, 281 µs total treatment time) and

stored in PET bottles had a shelf life of 21 days, whereas juice with the same treatment stored in

antimicrobial-incorporated PET bottles had a shelf life of 84 days.

Table 4. Effect of PEF on microbial inactivation.

Microorganisms Media PEF conditions

Log

Reduction

References

L. brevis

Orange

juice

35kv/cm, 4 µs, 32°C 5.8

Elez-Martínez

et al., 2005

S. cerevisiae

Apple

juice

20kv/cm, 10.4 pulses 4

Cserhalmi et

al., 2002

E. coli O157:H7

Apple

juice

34Kv/cm, 4µs, 38°C, 166 μs

treatment time

4.5

Evrendilek et

al., 2000

Z. bailli

Cranber

ry juice

36.5Kv/cm, 3.3 µs, 22°C 4.6

Raso et al.,

1998a

23

Z. bailli

Grape

juice

35Kv/cm, 2.3 µs, 20°C 5

Raso et al.,

1998a

B. fulva

conidiospores

Orange

juice

34.3 kV/cm, 2 µs 3.7

Raso et al.,

1998a

Total colony count/

Yeast and mold

Cactus

juice

35 kV/cm, 3 µs PW, 45 Hz

3 (Below

detectable

limit)

Moussa-

Ayoub et al.,

2017

E. coli (ATCC

11775)

Apple

juice

30.76 kV/cm, 40°C, 15

pulses, 21 μs treatment time

5

Moody et al.,

2014

4.2.3 Effect of PEF on physicochemical characteristics of juices

Many studies on pulsed electric field processing of fruit and vegetable juices have reported there

is no adverse effect of PEF on physicochemical characteristics, and in some cases, there is an

increase in the content of some bioactive compounds (Table 5). The retention of quality attributes

after treatment makes this technology superior to conventional thermal processing. Apple juice

treated by PEF has been shown to have no changes in sugars, ascorbic acid, or sensory

characteristics relative to untreated juice (Simpson et al, 1995). Some studies show that total

phenolic content in fruit juice increases with increase in energy input. Agcam et al. (2014) found

a higher total phenolic content in PEF-processed orange juice treated with higher energy (51.32 J)

compared to juice treated with lower energy (10.89 J). Extraction of the intracellular component

from pulp due to electroporation in the cell membrane must have contributed to the release of the

24

phenolic compound after PEF treatment; it is evident that higher energy input leads to a

correspondingly higher number of pores.

Leong et al. (2015) studied changes in the bioactive compounds of grape juice after PEF

treatment (1.5 kV/cm, 20 µs pulse width, 50 Hz, and 243/1033 pulses). The authors found that

PEF treatments increase the total vitamin C, anthocyanin content, phenolic compounds, and

antioxidant capacity in grape juice. The same authors also observed that higher pulse numbers can

enhance extraction of anthocyanin, phenolic compounds, and antioxidants better than low pulse

numbers, but observed the opposite result for vitamin C. Degradation of heat-sensitive vitamin C

might be due to the increased generation of heat over a higher number of pulses.

Table 5. Effect of PEF on physicochemical characteristics of juice.

Bioactive

compounds

Media PEF conditions

Retention

(%)

References

Total carotenoids

Orange

juice

35 kV/cm, 750 µs 90.67

Sánchez-

Moreno et al.,

2005

L-ascorbic acid

Orange

juice

35 kV/cm, 750 µs 93.01

Sánchez-

Moreno et al.,

2005

Total flavanones

Orange

juice

400 MPa, 1 min, 40°C >100

Sánchez-

Moreno et al.,

2005

25

Beta carotene

Fresh

orange-

carrot

blend

25-40 kV/cm, 60 µs >100

Torregrosa et

al., 2001

Total phenolic

compounds

Orange

juice

25.26 kV/cm, 1206 µs

treatment time

>100

Agcam et al.,

2014

Total anthocyanin

Blueberry

juice

5 kV/cm, 10 kJ/kg

>100 (60%

increase)

Bobinaitė et al.,

2015

Anthocyanin

(malvidin-3-O-

glucoside)

Grape

juice

1.5 kV/cm, 20 μs pulse

width, 50 Hz, 1033 pulses

>100

(146%

increase)

Leong et al.,

2016

Ascorbic acid

Orange-

Milk

beverage

15 kV/cm, 40 µs 34.5°C

97.6 %

retention

Zulueta et al.,

2010

Lycopene

Tomato

juice

35 kV/cm, 4 µs PW, 100

Hz

9.05

increase

Odriozola-

Serrano et al.,

2009

Beta carotene

Tomato

juice

35 kV/cm, 4 µs PW, 100

Hz

27.5

increase

Odriozola-

Serrano et al.,

2010

26

4.3 Power ultrasound

Ultrasound processing is a relatively new technique for pasteurizing liquid food. This technology

has been adopted for extraction of bioactive compounds from food, but it has also shown promising

results in microbial inactivation. The combination of ultrasound with other hurdles has proved to

be very effective in microbial inactivation; ultrasound can be effectively combined with pressure,

temperature, and antimicrobials. Thermo-sonication combines the effects of ultrasound and heat,

resulting in better inactivation than the use of heat alone. Low processing times, higher throughput,

and low energy consumption are some advantages of ultrasound over conventional thermal

processing (Zenker et al., 2003).

4.3.1 Power ultrasound and how it works

In ultrasound processing, transducers of an ultrasound device convert electrical energy to sound

energy; this sound energy is responsible for the production of ultrasonic waves. When these

ultrasonic waves propagate in a liquid medium, small bubbles form and collapse thousands of

times per second (Figure 3). This phenomenon is called cavitation, and is characterized by a series

of compression and rarefaction cycles (Sala et al., 1995; Feng, 2008; Soria and Villamiel, 2010).

Over time there will be an increase in the bubbles’ size characterized by regular shocks between

molecules, which are responsible for the rise of both temperature and pressure (Sala et al., 1995).

Cavitation generates microcurrents which can catalyze chemical reactions and disrupt microbial

cells, leading to the formation of wrinkles, ruptures, and perforation, ultimately causing cell death

(Barbosa-Cánovas and Rodríguez, 2002; Ugarte-Romero et al., 2006).

27

Figure 3. Cavitation in ultrasound (a) Bubble formation; (b) Bubble growth; (c) Bubble collapse (Source:

Abdullah and Chin, 2014).

4.3.2 Microbiological inactivation by ultrasound

Microbial inactivation by ultrasound depends on the types of microorganism present, the chosen

processing parameters, and the type of medium (Cheng et al., 2007). Inactivation is mainly

attributed to thinning of cell membranes, rise in temperature, generation of microcurrents, and

development of pressure gradient. The production of free radicals during cavitation is a minor

additional factor responsible for the lethal effect of US (Gould, 1995).

The efficacy of US treatment depends on the features of the targeted microbes. For

example, the cell membranes of gram-positive bacteria are more rigid than those of gram-negative

bacteria; this rigidity makes them more vulnerable to US. Aerobic microorganisms are more

susceptible than anaerobic species, and spores are the most resistant to this treatment (Gould, 1995;

Manas and Pagán, 2004). US has been shown to be less effective in spore reduction; ultrasound

treatment of 600 W, 20 kHz, 44°C, 95.2 µm for 30 min was unable to achieve any inactivation

28

of A. acidoterrestris spores in apple juice, while under the same conditions, there was a 2.8 log

reduction in S. cerevisiae (Ferrario et al., 2015).

As it is well-known, many parameters and their combinations affect the degree of

cavitation. Thus, according to Adekunte et al. (2010), when it comes to yeast, which has more rigid

cells compared to bacteria, inactivation rates are enhanced at higher ultrasound intensities. Based

on Moody et al. (2013), pulsed treatment is considered to be more efficient over short terms (15

min) than continuous treatment at 50ºC; however, after this period, both treatments reach the same

log reduction. An increase of 10ºC in the processing temperature could accomplish complete

inactivation in 5 min, regardless the treatment mode, either continuous or pulsed. Salleh-Mack and

Roberts (2007) state that pasteurization of juice that is higher in soluble solids requires a longer

treatment time to achieve 5 log reductions, as sugar and salt have some protective effects. To

minimize the effect of high solute concentrations, an increase in temperature during processing

should be considered.

The use of other preservation factors such as pH, antimicrobials, and temperature may act

synergistically with ultrasound to inactivate microorganisms (Table 6). When ultrasound is used

at mild temperature, efficient inactivation can be achieved by the combined effect of ultrasound

and heat; it shows a synergistic effect in microbial load reduction (Piyasena et al., 2003). Wordon

et al. (2012) found a significant decrease in the D-value of S. cerevisiae by ultrasound processing

at 60°C (D-value 0.73 min) compared to treatment with temperature alone (D-value 3.53 min) and

ultrasound alone (D-value 3.1 min). Ultrasound combined with mild temperature is effective

against foodborne pathogens including E. coli (Salleh-Mack and Roberts, 2007). Lee et al. (2009)

studied the inactivation of E. coli K12 using ultrasound combined with heat and/or pressurization,

29

and observed that combination of US with increased temperature and/or pressure significantly

reduced the time required to achieve a 5 log reduction.

Table 6. Microbial inactivation in juices by Ultrasound.

Microorganisms Media Ultrasound conditions

Log

Reduction

References

Saccharomyces

cerevisiae

Grape

juice

24 kHz, 120 μm, 60°C,

10 min

7

Bermúdez-Aguirre and

Barbosa-Cánovas,

2012

E. coli (ATCC

11775)

Apple

juice

24 kHz, 120 μm, 60°C,

5 min

7 (complete

inactivation)

Moody et al., 2014

E. coli 0157:H7

Blueberry

juice

700 W, 60 °C, 10 min 5.1 Zhu et al., 2017

Saccharomyces

cerevisiae

Apple

juice

600 W, 20 kHz, 44°C,

95.2 μm

2.8 Ferrario et al., 2015

Aerobic

mesophilic count

Orange

juice

20 kHz, 89.25 µm, 8

min

1.38

Gómez-López et al.,

2010

Total plate count

and

Enterobacteria

Cactus

pear juice

1500 W, 20 kHz, 80%

amplitude, 63.4C 15

min,

4 (Below the

detection

limit)

Zafra-Rojas et al.,

2013

E. coli K12

Apple

cider

20 kHz, 3 W/mL, 3.8

min, 59°C

5 Lee et al., 2013

30

L.

monocytogenes

0.85%

NaCl

22.3 kHz, 1.43 W/mL,

10 min, 37°C

5

Ugarte-Romero et al.,

2007

4.3.3 Effect of ultrasound on physicochemical characteristics of juices

Ultrasound treatment has shown to preserve the physicochemical qualities of treated juice (Table

7). These effects may be related to cavitation-promoted degassing removing oxygen from the

medium (Bermudez-Aguirre and Barbosa-Cánovas, 2012). Some controversy remains on this

point due to factors including food matrix diversity, and differences in processing conditions and

energy input.

Aadil et al. (2013) reported a significant increase in the ascorbic acid content of apple juice

after sonication for 60 or 90 min; treatment for shorter times had no significant effect on its

ascorbic acid content. In contrast, Santhirasegaram et al. (2013) found that mango juice processed

either by sonication or thermal treatment had a significantly decreased ascorbic acid content

compared to an untreated control. Thus, it is important to consider that the food matrix, as well as

the processing parameters, has a significant influence on the final product obtained. Studies

conducted by Cheng et al. (2007) has observed that samples of guava juice after carbonation and/or

sonication show the highest percentage of ascorbic acid. In that study, researchers incorporated

dry ice during carbonation, which leads to a decline in temperature, preventing ascorbic acid

degradation (a temperature-dependent reaction). Likewise, sonication can remove dissolved

oxygen, responsible for catalyzing another degradation pathway, from the medium.

Abid et al. (2013) also observed a significant increase in total phenolic content in sonicated

apple juice treated for 60 and 90 min. Similarly, Santhirasegaram et al. (2013) and Bhat et al.

31

(2011) respectively, in their studies of sonicated mango juice and kasturi lime juice found

enhancement in the content of phenolic compounds when sonication was applied. The increase in

the percentage of the phenolic compound during sonication might be related to disruption of

biological cell walls, promoting the release of cell contents due to cavitational collapse around

colloidal particles. This outcome could also be caused by reactions between hydroxyl radicals

generated during sonication and the aromatic ring of polyphenols (Ashokkumar et al., 2008; Zafra-

Rojas et al., 2013). A slump in the phenolic content could take place once cavitational bubbles

filled with water vapor or gases (such as oxygen) dissolve in the fluid; their inclusion might

contribute to the oxidative degradation of phenolic compounds. Therefore, these results emphasize

that food matrix and processing conditions are important for sonication performance (Fonteles et

al., 2012).

Retention of phenolic compounds also depends on the processing time. Rawson et al.

(2011) found that processing times <6 min did not have much effect on phenol degradation, but

over longer processing times higher degradation was observed. For example, in the US processing

of watermelon juice a treatment time of 2 min yielded 83% retention of phenolic compounds, and

after a processing time of 10 min only 42% retention was observed.

Previous studies have shown an increase (P<0.05) in the carotenoid content of apple juice

after sonication treatments of 30 and 60 min, possibly due to mechanical disruption of cell walls,

but in at least one study, sonication did not reveal significant (P>0.05) differences as compared to

untreated juice samples (Abid et al., 2014).

Many studies on the effect of ultrasound on anthocyanin show that there is either 100%

retention or an increase in the content of free anthocyanin. A study conducted by Golmohamadi et

32

al. (2013) indicates that the total monomeric anthocyanin content of red raspberry puree rose by

12.6% after ultrasound treatment (400 W, 20 kHz, 10 min); this increment in ultrasound decreases

with increase in frequency. The same authors found a 6.7% increase in total monomeric

anthocyanin content when the frequency was increased from 20 kHz to 490 kHz.

Table 7. Effect of ultrasound on juice physiochemical characteristics.

Bioactive

compounds

Media Ultrasound conditions

Retention

(%)

References

Ascorbic acid

Carrot

juice

20 kHz, 100 W, 15 min,

<30°C

94

Khandpur et al.,

2015

Ascorbic acid

Tomato

juice

20 kHz, 61 µm, 10 min,

32-45°C, 10 min

67

Adekunte et al.,

2010

Total phenolic Pear juice

20 kHz, 750 W, 65°C,

10 min

94

Saeeduddin et al.,

2015

Antioxidant

capacity

Pear juice

21 kHz, 750 W, 65°C,

10 min

86

Saeeduddin et al.,

2015

Chelating activity

Cactus

pear juice

20 kHz, 1500 W, 80%

amplitude, 25 min

67

Zafra-Rojas et al.,

2013

Ascorbic acid

Orange

juice

1500 W, 20 kHz, 25°C,

10 min

95 Tiwari et al., 2009

Total monomeric

anthocyanin

Red

raspberry

puree

20 kHz, 10 min

>100 (12.6%

increase)

Golmohamadi et

al., 2013

33

Total phenolic

content

Water

melon

juice

1500 W, 20 kHz, 61 μm,

2 min

83.23

Rawson et al.,

2011

Total flavonoids

Apple

juice

25 kHz, 20ºC, 70%

Amplitude, 30 min

>100 (4.22

% increase)

Abid et al., 2013

4.4 Combination of processes

Several of these treatments can stand alone, but their use in various combinations has been

investigated, and is often more effective. As has been alluded in earlier sections, synergy has been

shown between a variety of factors, including pressure, temperature, ultrasound, and addition of

antimicrobial agents such as nisin. A discussion of some combinations of those technologies

follows.

4.4.1 Ultrasound and high pressure processing

The combination of ultrasound and high pressure processing has shown promising results for

microbial inactivation in fresh liquid products (Feng, 2011). The schematic diagram of this type

of combination is shown in Figure 4. This combination, mano-sonication (MS), uses ultrasound at

moderate pressures, lower than those commonly used in high pressure processing (<600 kPa).

When these treatments are combined, processing can occur at room temperature. When ultrasound

is used without pressure, processing at room temperature is not very effective (Raso et al., 1998b).

Pagán et al. (1999a) observed a significant increase in the inactivation rate of L.

monocytogenes when ultrasound was combined with a pressure of 200 kPa as compared to ambient

34

pressure. As the cavitation phenomenon is intensified at higher pressure the effectiveness of

ultrasound processing increases with an increase in pressure. There are very few studies of the

synergistic effect of combining ultrasound and pressure. Feng et al. (2011) found no synergistic

effect of these two parameters, but did find an additive effect (Pagán et al., 1999b). Although no

synergism was observed in the combination of ultrasound and high pressure, synergisms was

observed in the case of combining ultrasound, pressure, and temperature (Feng et al., 2011,

Demirci and Ngadi, 2012).

Application of pressure with ultrasound significantly reduces the processing time required

to inactive microorganisms. Ultrasound alone is not very effective, and pressure alone is a

comparatively expensive process due the equipment and operating costs to maintain high pressure

(>400 MPa). Their combination, however, can be industrialized for lower-cost pasteurization of

fresh juice, with the added advantage of increased extraction of intracellular components. The

combination can also prevent the separation of binary juice blends in the bottles after processing,

as ultrasound homogenizes and HPP pasteurizes.

35

Figure 4. Combination of ultrasound and pressure (Source: Lee et al., 2009).

4.4.2 Ultrasound and pulsed electric fields

Ultrasound is a very flexible technology; it can be used for different purposes. Ultrasound can be

used in milk homogenization, and the homogenized milk can be pasteurized by pulsed electric

field. The microbial load will be reduced by ultrasound as well, and the remaining load will be

inactivated by PEF. Ultrasound injures microbial cells during cavitation, making them vulnerable

to inactivation by PEF. Furthermore, US-homogenized milk is easier to process by PEF as the size

of the fat globules is reduced allowing smooth flow in the PEF chamber with less chance of sparks

due to dielectric breakdown.

Combining PEF and mano-thermo-sonication for inactivation of Listeria innocua caused

only an additive effect, as the sum of individual effects was not significantly (p>0.05) different

than the combined effect (Palgan et al., 2012). Although no synergism was observed in the

36

microbial inactivation by combining PEF and ultrasound, this combination could result in

synergism in the extraction of bioactive compounds from plant cells, as ultrasound and PEF break

down cells by different mechanisms, which could result in higher extraction. Synergism in the

combination of these technologies could potentially be achieved by the incorporation of other

stress factors, and the combination of these technologies has industrial potentials in

homogenization of milk as well as in the extraction of bioactive compounds.

5. Final remarks

Nonthermal technologies such as HPP, PEF, and US have proven to be effective methods for

microbial load reduction in fruit and vegetable juices with minimal or no impact on bioactive

compounds. Of the three technologies discussed, HPP is already commercialized in the food and

beverage processing industries. PEF has been used at the industrial level for juice processing as

well as for extraction of bioactive compounds and to create pores in tissues in order to facilitate

other processes such as dehydration. Microbial inactivation in juice by PEF can be further

enhanced by the incorporation of other stress factors making the overall process quite efficient.

Ultrasound technology has not yet been commercialized for juice pasteurization purposes,

although it has been used in applications including extraction of intracellular components and pre-

treatment.

Even though HPP and PEF are commercialized in the food industry, there is still much to

do to make them more efficient, i.e. to reduce processing costs and increase throughput. PEF and

ultrasound applied alone often may not lead to the desired levels of inactivation in some juices.

For this reason, in some cases, these technologies need to be combined with other hurdles such as

mild temperature or addition of natural antimicrobials. Synergistic inactivation of microorganisms

37

can be achieved when these technologies are combined with other hurdles. For example,

incorporation of mild temperature with PEF could result in desired reductions of target

microorganisms at sub-lethal temperatures in fractions of a second, whereas temperature alone or

PEF alone could not render these results. More studies need to be carried out on the combination

of HPP, PEF, or US on the processing of juices. The effect of antimicrobials at different

temperatures is another area where it needs in-depth study for the processing of juices.

Validation studies need to be carried out to determine the feasibility of combining these

nonthermal technologies with other hurdles on an industrial scale. There is great potential in the

beverage processing industry to develop premium products with enhanced nutritional benefits by

applying these nonthermal processing methods.

38

References

Aadil, R. M., Zeng, X., Han, Z., & Sun, D. (2013). Effects of ultrasound treatments on quality of

grapefruit juice. Food Chemistry, 141(3), 3201-3206.

Abdullah, N., & Chin, N. L. (2014). Application of thermosonication treatment in processing and

production of high quality and safe-to-drink fruit juices. Agriculture and Agricultural

Science Procedia, 2, 320-327.

Abid, M., Jabbar, S., Wu, T., Hashim, M. M., Hu, B., Lei, S. & Zeng, X (2014). Sonication

enhances polyphenolic compounds, sugars, carotenoids and mineral elements of apple

juice. Ultrasonics Sonochemistry, 21, 93-97.

Abid, M., Jabbar, S., Wu, T., Hashim, M. M., Hu, B., Lei, S., ... & Zeng, X. (2013). Effect of

ultrasound on different quality parameters of apple juice. Ultrasonics

Sonochemistry, 20(5), 1182-1187.

Adekunte, A. O., Tiwari, B. K., Cullen, P. J., Scannell, A. G. M., & O’Donnell, C. P. (2010). Effect

of sonication on colour, ascorbic acid and yeast inactivation in tomato juice. Food

Chemistry, 122(3), 500-507.

Agcam, E., Akyıldız, A., & Evrendilek, G. A. (2014). Comparison of phenolic compounds of

orange juice processed by pulsed electric fields (PEF) and conventional thermal

pasteurisation. Food Chemistry, 143, 354-361.

Alpas, H. (2013). Effect of high hydrostatic pressure processing (HHP) on quality properties,

squeezing pressure effect and shelf life of pomegranate juice. Current Opinion in

Biotechnology, (24), S111.

39

Altunakar, B. (2007). Food Preservation by Pulsed Electric Fields and Selected Antimicrobials.

(Doctoral dissertation, Washington State University, USA).

Andrés, V., Villanueva, M. J., & Tenorio, M. D. (2016). The effect of high-pressure processing on

colour, bioactive compounds, and antioxidant activity in smoothies during refrigerated

storage. Food Chemistry, 192, 328-335.

Ashokkumar, M., Sunartio, D., Kentish, S., Mawson, R., Simons, L., Vilkhu, K., & Versteeg, C.

K. (2008). Modification of food ingredients by ultrasound to improve functionality: a

preliminary study on a model system. Innovative Food Science and Emerging

Technologies, 9(2), 155-160.

Barba, F. J., Esteve, M. J., & Frigola, A. (2013). Physicochemical and nutritional characteristics

of blueberry juice after high pressure processing. Food Research International, 50(2), 545-

549.

Barbosa-Cánovas, G. V., & Rodríguez, J. J. (2002). Update on nonthermal food processing

technologies: Pulsed electric field, high hydrostatic pressure, irradiation and ultrasound.

Food Australia, 54(11), 513-520.

Bermúdez-Aguirre, D., & Barbosa-Cánovas, G. V. (2012). Inactivation of Saccharomyces

cerevisiae in pineapple, grape and cranberry juices under pulsed and continuous thermo-

sonication treatments. Journal of Food Engineering, 108(3), 383-392.

Bhat, R., Kamaruddin, N. S. B. C., Min-Tze, L., & Karim, A. A. (2011). Sonication improves

kasturi lime (Citrus microcarpa) juice quality. Ultrasonics Sonochemistry, 18(6), 1295-

1300.

40

Bobinaitė, R., Pataro, G., Lamanauskas, N., Šatkauskas, S., Viškelis, P., & Ferrari, G. (2015).

Application of pulsed electric field in the production of juice and extraction of bioactive

compounds from blueberry fruits and their by-products. Journal of Food Science and

Technology, 52(9), 5898-5905.

Buzrul, S., Alpas, H., Largeteau, A., & Demazeau, G. (2008). Inactivation of Escherichia coli and

Listeria innocua in kiwifruit and pineapple juices by high hydrostatic

pressure. International Journal of Food Microbiology, 124(3), 275-278.

CDC. (2015). Foodborne Outbreak Online Database (FOOD).

http://wwwn.cdc.gov/foodborneoutbreaks/Default.aspx. Accessed February 4, 2016

Chaikham, P., & Apichartsrangkoon, A. (2012). Comparison of dynamic viscoelastic and

physicochemical properties of pressurised and pasteurised longan juices with xanthan

addition. Food Chemistry, 134(4), 2194-2200.

Chen, D., Xi, H., Guo, X., Qin, Z., Pang, X., Hu, X., ... & Wu, J. (2013). Comparative study of

quality of cloudy pomegranate juice treated by high hydrostatic pressure and high

temperature short time. Innovative Food Science and Emerging Technologies, 19, 85-94.

Cheng, L. H., Soh, C. Y., Liew, S. C., & Teh, F. F. (2007). Effects of sonication and carbonation

on guava juice quality. Food Chemistry, 104(4), 1396-1401.

Cheng, L., Soh, C., Liew, S., & Teh, F. (2007). Effects of sonication and carbonation on guava

juice quality. Food Chemistry, 104(4), 1396-1401.

41

Chung, H. J., & Yousef, A. E. (2010). Synergistic effect of high pressure processing and

Lactobacillus casei antimicrobial activity against pressure resistant Listeria

monocytogenes. New Biotechnology, 27(4), 403-408.

Cserhalmi, Z., Vidács, I., Beczner, J., & Czukor, B. (2002). Inactivation of Saccharomyces

cerevisiae and Bacillus cereus by pulsed electric fields technology. Innovative Food

Science and Emerging Technologies, 3(1), 41-45.

D’Amico, D. J., Silk, T. M., Wu, J. and Guo, M. (2006). Inactivation of microorganisms in milk

and apple cider treated with ultrasound. Journal of Food Protection, 69, 556–563.

da Rocha Ferreira, E. H., Rosenthal, A., Calado, V., Saraiva, J., & Mendo, S. (2009).

Byssochlamys nivea inactivation in pineapple juice and nectar using high pressure

cycles. Journal of Food Engineering, 95(4), 664-669.

Demirci, A., & Ngadi, M. O. (Eds.). (2012). Microbial decontamination in the food industry:

Novel methods and applications. Elsevier.

Elez-Martinez, P., Escolà-Hernández, J., Soliva-Fortuny, R. C., & Martín-Belloso, O. (2005).

Inactivation of Lactobacillus brevis in orange juice by high-intensity pulsed electric fields.

Food Microbiology, 22(4), 311-319.

Evrendilek, G. A., Jin, Z. T., Ruhlman, K. T., Qiu, X., Zhang, Q. H., & Richter, E. R. (2000).

Microbial safety and shelf-life of apple juice and cider processed by bench and pilot scale

PEF systems. Innovative Food Science and Emerging Technologies, 1(1), 77-86.

Feng H, Yang W, Hielscher T. (2008). Power ultrasound. Food Science and Technology

International, 14, 433-436.

42

Feng, H., Barbosa-Cánovas, G. V., & Weiss, J. (2011). Ultrasound technologies for food and

bioprocessing (Vol. 1, p. 599). New York: Springer.

Fernández-Sestelo, A., de Saá, R. S., Pérez-Lamela, C., Torrado-Agrasar, A., Rúa, M. L., &

Pastrana-Castro, L. (2013). Overall quality properties in pressurized kiwi purée: Microbial,

physicochemical, nutritive and sensory tests during refrigerated storage. Innovative Food

Science and Emerging Technologies, 20, 64-72.

Ferrari, G., Maresca, P., & Ciccarone, R. (2011). The effects of high hydrostatic pressure on the

polyphenols and anthocyanins in red fruit products. Procedia Food Science, 1, 847-853.

Ferrario, M., Alzamora, S. M., & Guerrero, S. (2015). Study of the inactivation of spoilage

microorganisms in apple juice by pulsed light and ultrasound. Food Microbiology, 46, 635-

642.

Fonteles, T. V., Costa, M. G., Jesus, A. L., Miranda, M. R., Fernandes, F. A., & Rodrigues, S.

(2012). Power ultrasound processing of cantaloupe melon juice: Effects on quality

parameters. Food Research International, 48(1), 41-48.

Golmohamadi, A., Möller, G., Powers, J., & Nindo, C. (2013). Effect of ultrasound frequency on

antioxidant activity, total phenolic and anthocyanin content of red raspberry

puree. Ultrasonics Sonochemistry, 20(5), 1316-1323.

Gómez-López, V. M., Orsolani, L., Martínez-Yépez, A., & Tapia, M. S. (2010). Microbiological

and sensory quality of sonicated calcium-added orange juice. LWT-Food Science and

Technology, 43(5), 808-813.

43

Gould, G. W (1995). New Methods of Food Preservation. Unilever Research Laboratory, 176-

190.

Hager, T. J., Howard, L. R., & Prior, R. L. (2008). Processing and storage effects on monomeric

anthocyanins, percent polymeric color, and antioxidant capacity of processed blackberry

products. Journal of Agricultural and Food Chemistry, 56(3), 689-695.

Hiremath, N. D., & Ramaswamy, H. S. (2012). High‐pressure destruction kinetics of spoilage and

pathogenic microorganisms in mango juice. Journal of Food Processing and

Preservation, 36(2), 113-125.

Jain, S., Bidol, S. A., Austin, J. L., Berl, E., Elson, F., Williams, M. L., ... & Yu, P. A. (2009).

Multistate outbreak of Salmonella Typhimurium and Saintpaul infections associated with

unpasteurized orange juice—United States, 2005. Clinical Infectious Diseases, 48(8),

1065-1071.

Jin, T.Z., Guo, M., and Yang, R. (2014). Combination of pulsed electric field processing and

antimicrobial bottle for extending microbiological shelf-life of pomegranate juice.

Innovative Food Science and Emerging Technologies, 26, 153-158.

Kaşikçi, M. B., & Bağdatlioğlu, N. (2016). High hydrostatic pressure treatment of fruit, fruit

products and fruit juices: a review on phenolic compounds. Journal of Food and Health

Science, 2(1), 27-39.

Khandpur, P., & Gogate, P. R. (2015). Effect of novel ultrasound based processing on the nutrition

quality of different fruit and vegetable juices. Ultrasonics Sonochemistry, 27, 125-136.

44

Knorr, D., Froehling, A., Jaeger, H., Reineke, K., Schlueter, O., & Schoessler, K. (2011a).

Emerging technologies in food processing. Annual Review of Food Science and

Technology, 2, 203-235.

Knorr, D., Reineke, K., Mathys, A., Heinz, V., and Buckow, R. (2011b). High-pressure-induced

effects on bacterial spores, vegetative microorganisms, and enzymes. In Food Engineering

Interfaces (pp. 325-340). Springer New York.

Lado, B. H., & Yousef, A. E. (2002). Alternative food-preservation technologies: efficacy and

mechanisms. Microbes and Infection, 4(4), 433-440.

Lee, H., Kim, H., Cadwallader, K. R., Feng, H., & Martin, S. E. (2013). Sonication in combination

with heat and low pressure as an alternative pasteurization treatment–Effect on Escherichia

coli K12 inactivation and quality of apple cider. Ultrasonics Sonochemistry, 20(4), 1131-

1138.

Lee, H., Zhou, B., Feng, H., & Martin, S. E. (2009). Effect of pH on inactivation of Escherichia

coli K12 by sonication, manosonication, thermosonication, and manothermosonication.

Journal of Food Science, 74(4), 191-198.

Lee, H., Zhou, B., Liang, W., Feng, H., & Martin, S. E. (2009). Inactivation of Escherichia coli

cells with sonication, manosonication, thermosonication, and manothermosonication:

microbial responses and kinetics modeling. Journal of Food Engineering, 93(3), 354-364.

Leong, S. Y., Burritt, D. J., & Oey, I. (2016). Evaluation of the anthocyanin release and health-

promoting properties of Pinot Noir grape juices after pulsed electric fields. Food

Chemistry, 196, 833-841.

45

Liang, Z., Mittal, G.S., and Griffiths, M. W. (2002). Inactivation of Salmonella Typhimurium in

orange juice containing antimicrobial agents by pulsed electric field. Journal of Food

Protection, 65(7), 1081-1087.

Liu, F., Wang, Y., Li, R., Bi, X., & Liao, X. (2014). Effects of high hydrostatic pressure and high

temperature short time on antioxidant activity, antioxidant compounds and color of mango

nectars. Innovative Food Science and Emerging Technologies, 21, 35-43.

Manas, P., & Pagán, R. (2005). Microbial inactivation by new technologies of food

preservation. Journal of Applied Microbiology, 98(6), 1387-1399.

Margosch, D. (2005). Behavior of bacterial endospores and toxins as safety determinants in low

acid pressurized food. (Doctoral dissertation, Technical University Berlin, Germany).

McNamee, C., Noci, F., Cronin, D. A., Lyng, J. G., Morgan, D. J., & Scannell, A. G. M. (2010).

PEF based hurdle strategy to control Pichia fermentans, Listeria innocua and Escherichia

coli K12 in orange juice. International Journal of Food Microbiology, 138(1), 13-18.

Moody, A., Marx, G., Swanson, B. G., & Bermúdez-Aguirre, D. (2014). A comprehensive study

on the inactivation of Escherichia coli under nonthermal technologies: High hydrostatic

pressure, pulsed electric fields and ultrasound. Food Control, 37, 305-314.

Mosqueda-Melgar, J., Raybaudi-Massilia, R. M., & Martin-Belloso, O. (2008). Combination of

high-intensity pulsed electric fields with natural antimicrobials to inactivate pathogenic

microorganisms and extend the shelf-life of melon and watermelon juices. Food

Microbiology, 25(3), 479-491.

46

Moussa-Ayoub, T. E., Jäger, H., Knorr, D., El-Samahy, S. K., Kroh, L. W., & Rohn, S. (2017).

Impact of pulsed electric fields, high hydrostatic pressure, and thermal pasteurization on

selected characteristics of Opuntia dillenii cactus juice. LWT-Food Science and

Technology, 79, 534-542.

Munoz, M., de Ancos, Begoña., & Cano, M. P. (2007). Effects of high pressure and mild heat on

endogenous microflora and on the inactivation and sublethal injury of Escherichia coli

inoculated into fruit juices and vegetable soup. Journal of Food Protection, 70(7), 1587-

1593.

Nguyen, P., and Mittal, G.S. (2007). Inactivation of naturally occurring microorganisms in tomato

juice using pulsed electric field (PEF) with and without antimicrobials. Chemical

Engineering and Processing: Process Intensification, 46(4), 360-365.

Odriozola-Serrano, I., Soliva-Fortuny, R., Hernández-Jover, T., & Martín-Belloso, O. (2009).

Carotenoid and phenolic profile of tomato juices processed by high intensity pulsed electric

fields compared with conventional thermal treatments. Food Chemistry, 112(1), 258-266.

Pagán, R., Manas, P., Alvarez, I., & Condón, S. (1999a). Resistance of Listeria monocytogenes to

ultrasonic waves under pressure at sublethal (manosonication) and lethal

(manothermosonication). Food Microbiology, 16(2), 139-148.

Pagán, R., Manas, P., Raso, J., & Condón, S. (1999b). Bacterial resistance to ultrasonic waves

under pressure at nonlethal (manosonication) and lethal (manothermosonication)

temperatures. Applied and Environmental Microbiology, 65(1), 297-300.

Patras, A., Brunton, N. P., Da Pieve, S., & Butler, F. (2009). Impact of high pressure processing

on total antioxidant activity, phenolic, ascorbic acid, anthocyanin content and colour of

47

strawberry and blackberry purées. Innovative Food Science and Emerging


Patterson, M. F., McKay, A. M., Connolly, M., & Linton, M. (2012). The effect of high hydrostatic

pressure on the microbiological quality and safety of carrot juice during refrigerated

storage. Food Microbiology, 30(1), 205-212.

Pilavtepe‐Çelik, M. (2013). High hydrostatic pressure (HHP) inactivation of foodborne pathogens

in low‐acid juices. International Journal of Food Science & Technology, 48(4), 673-677.

Piyasena, P., Mohareb, E., & McKellar, R. C. (2003). Inactivation of microbes using ultrasound:

a review. International Journal of Food Microbiology, 87(3), 207-216.

Raso, J., Calderón, M. L., Góngora, M., Barbosa‐Cánovas, G. V., & Swanson, B. G. (1998a).

Inactivation of Zygosaccharomyces bailii in fruit juices by heat, high hydrostatic pressure

and pulsed electric fields. Journal of Food Science, 63(6), 1042-1044.

Raso, J., Pagan, R., Condon, S., & Sala, F. J. (1998b). Influence of temperature and pressure on

the lethality of ultrasound. Applied and Environmental Microbiology, 64(2), 465-471.

Rawson, A., Patras, A., Tiwari, B. K., Noci, F., Koutchma, T., & Brunton, N. (2011). Effect of

thermal and nonthermal processing technologies on the bioactive content of exotic fruits

and their products: Review of recent advances. Food Research International, 44(7), 1875-

1887.

Rawson, A., Tiwari, B. K., Patras, A., Brunton, N., Brennan, C., Cullen, P. J., & O'Donnell, C.

(2011). Effect of thermosonication on bioactive compounds in watermelon juice. Food

Research International, 44(5), 1168-1173.

48

Saeeduddin, M., Abid, M., Jabbar, S., Wu, T., Hashim, M. M., Awad, F. N., ... & Zeng, X. (2015).

Quality assessment of pear juice under ultrasound and commercial pasteurization

processing conditions. LWT-Food Science and Technology, 64(1), 452-458.

Sala, F. J., Burgos, J., Condon, S., Lopez, P., & Raso, J. (1995). Effect of heat and ultrasound on

microorganisms and enzymes. In New Methods of Food Preservation (pp. 176-204).

Springer US.

Sánchez-Moreno, C., Plaza, L., Elez-Martínez, P., De Ancos, B., Martín-Belloso, O., & Cano, M.

P. (2005). Impact of high pressure and pulsed electric fields on bioactive compounds and

antioxidant activity of orange juice in comparison with traditional thermal

processing. Journal of Agricultural and Food Chemistry, 53(11), 4403-4409.

Santhirasegaram, V., Razali, Z., & Somasundram, C. (2013). Effects of thermal treatment and

sonication on quality attributes of Chokanan mango (Mangifera indica L.)

juice. Ultrasonics Sonochemistry, 20(5), 1276-1282.

Shamsi, K. (2008). Effects of pulsed electric field processing on microbial, enzymatic and physical

attributes of milk and the rennet-induced milk gels. RMIT University, Melbourne, Australia

(December 2008, PhD thesis).

Simpson, M. V., Barbosa-Cánovas, G. V. and Swanson, B. G. (1995). The Combined inhibitory

effect of lysozyme and high voltage pulsed electric fields on the growth of Bacillus subtilis

spores. IFT Annual Meeting: Book of Abstracts. 267.

Sokołowska, B., Skąpska, S., Fonberg-Broczek, M., Niezgoda, J., Chotkiewicz, M., Dekowska,

A., and Rzoska, S. (2012). The combined effect of high pressure and nisin or lysozyme on

49

the inactivation of Alicyclobacillus acidoterrestris spores in apple juice. High Pressure

Research, 32(1), 119-127.

Soria, A. C., & Villamiel, M. (2010). Effect of ultrasound on the technological properties and

bioactivity of food: A review. Trends in Food Science and Technology, 21(7), 323-331.

Syed, Q. A., Buffa, M., Guamis, B., & Saldo, J. (2013). Lethality and injuring the effect of

compression and decompression rates of high hydrostatic pressure on Escherichia coli

O157:H7 in different matrices. High Pressure Research, 33(1), 64-72.

Syed, Q. A., Reineke, K., Saldo, J., Buffa, M., Guamis, B., & Knorr, D. (2012). Effect of

compression and decompression rates during high hydrostatic pressure processing on

inactivation kinetics of bacterial spores at different temperatures. Food Control, 25(1),

361-367.

Timmermans, R. A. H., Mastwijk, H. C., Knol, J. J., Quataert, M. C. J., Vervoort, L., Van der

Plancken, I., ... & Matser, A. M. (2011). Comparing equivalent thermal, high pressure and

pulsed electric field processes for mild pasteurization of orange juice. Part I: Impact on

overall quality attributes. Innovative Food Science & Emerging Technologies, 12(3), 235-

243.

Tiwari, B. K., O'Donnell, C. P., Muthukumarappan, K., & Cullen, P. J. (2009). Ascorbic acid

degradation kinetics of sonicated orange juice during storage and comparison with

thermally pasteurised juice. LWT-Food Science and Technology, 42(3), 700-704.

Tola, Y. B., & Ramaswamy, H. S. (2014). Combined effects of high pressure, moderate heat and

pH on the inactivation kinetics of Bacillus licheniformis spores in carrot juice. Food

Research International, 62, 50-58.

50

Torregrosa, F., Cortés, C., Esteve, M. J., & Frígola, A. (2005). Effect of high-intensity pulsed

electric fields processing and conventional heat treatment on orange− carrot juice

carotenoids. Journal of Agricultural and Food Chemistry, 53(24), 9519-9525.

Ugarte‐Romero, E., Feng, H., & Martin, S. E. (2007). Inactivation of Shigella boydii 18 IDPH and

Listeria monocytogenes Scott A with power ultrasound at different acoustic energy

densities and temperatures. Journal of Food Science, 72(4).

Ugarte‐Romero, E., Feng, H., Martin, S. E., Cadwallader, K. R., & Robinson, S. J. (2006).

Inactivation of Escherichia coli with power ultrasound in apple cider. Journal of Food

Science, 71(2).

Varela-Santos, E., Ochoa-Martinez, A., Tabilo-Munizaga, G., Reyes, J. E., Pérez-Won, M.,

Briones-Labarca, V., & Morales-Castro, J. (2012). Effect of high hydrostatic pressure

(HHP) processing on physicochemical properties, bioactive compounds and shelf-life of

pomegranate juice. Innovative Food Science and Emerging Technologies, 13, 13-22.

Voldřich, M., Dobiáš, J., Tichá, L., Čeřovský, M., & Krátká, J. (2004). Resistance of vegetative

cells and ascospores of heat resistant mould Talaromyces avellaneus to the high pressure

treatment in apple juice. Journal of Food Engineering, 61(4), 541-543.

Vega-Mercado, H., Pothakamury, U. R., Chang, F. J., Barbosa-Cánovas, G. V., & Swanson, B. G.

(1996). Inactivation of Escherichia coli by combining pH, ionic strength and pulsed electric

fields hurdles. Food Research International, 29(2), 117-121.

Weemaes, C. A., Ludikhuyze, L. R., Van den Broeck, I., Hendrickx, M. E., & Tobback, P. P.

(1998). Activity, electrophoretic characteristics and heat inactivation of

51

polyphenoloxidases from apples, avocados, grapes, pears and plums. LWT-Food Science


Winter, R., and Jeworrek, C. (2009). Effect of pressure on membranes. Soft Matter, 5(17), 3157-

3173.

Wolbang, C. M., Fitos, J. L., & Treeby, M. T. (2008). The effect of high pressure processing on

nutritional value and quality attributes of Cucumis melo L. Innovative Food Science and


Wordon, B. A., Mortimer, B., & McMaster, L. D. (2012). Comparative real-time analysis of

Saccharomyces cerevisiae cell viability, injury and death induced by ultrasound (20 kHz)

and heat for the application of hurdle technology. Food Research International, 47(2), 134-

139.

Wu, J. (2014). Eggs and egg products processing. Food Processing: Principles and Applications,

Second Edition, 437-455.

Xi, J., Shen, D., Zhao, S., Lu, B., Li, Y., & Zhang, R. (2009). Characterization of polyphenols

from green tea leaves using a high hydrostatic pressure extraction. International Journal

of Pharmaceutics, 382(1), 139-143.

Yu, Y., Xu, Y., Wu, J., Xiao, G., Fu, M., & Zhang, Y. (2014). Effect of ultra-high pressure

homogenisation processing on phenolic compounds, antioxidant capacity and anti-

glucosidase of mulberry juice. Food Chemistry, 153, 114-120.

52

Yuste, J., Capellas, M., Pla, R., Fung, D. Y., & Mor‐Mur, M. (2001). High pressure processing for

food safety and preservation: a review. Journal of Rapid Methods and Automation in


Zafra-Rojas, Q. Y., Cruz-Cansino, N., Ramírez-Moreno, E., Delgado-Olivares, L., Villanueva-

Sánchez, J., & Alanís-García, E. (2013). Effects of ultrasound treatment in purple cactus

pear (Opuntia ficus-indica) juice. Ultrasonics Sonochemistry, 20(5), 1283-1288.

Zenker, M., Heinz, V., & Knorr, D. (2003). Application of ultrasound-assisted thermal processing

for preservation and quality retention of liquid foods. Journal of Food Protection, 66(9),

1642-1649.

Zhang, Q. H., Barbosa-Cánovas, G. V., & Swanson, B. G. (1995). Engineering aspects of pulsed

electric field pasteurization. Journal of Food Engineering, 25, 261-281.

Zhu, J., Wang, Y., Li, X., Li, B., Liu, S., Chang, N., ... & Meng, X. (2017). Combined effect of

ultrasound, heat, and pressure on Escherichia coli O157:H7, polyphenol oxidase activity,

and anthocyanins in blueberry (Vaccinium corymbosum) juice. Ultrasonics

Sonochemistry, 37, 251-259.

Zimmermann, M., Schaffner, D. W., & Aragão, G. M. (2013). Modeling the inactivation kinetics

of Bacillus coagulans spores in tomato pulp from the combined effect of high pressure and

moderate temperature. LWT-Food Science and Technology, 53(1), 107-112.

Zimmermann, U. (1986) Electrical breakdown, electropermeabilization and electrofusion. Reviews

of Physiology, Biochemistry and Pharmacology 105, 175-256.

53

Zulueta, A., Esteve, M. J., & Frígola, A. (2010). Ascorbic acid in orange juice–milk beverage

treated by high intensity pulsed electric fields and its stability during storage. Innovative

Food Science and Emerging Technologies, 11(1), 84-90.

54

CHAPTER TWO

ENGINEERING ASPECTS OF NOVEL TECHNOLOGIES FOR BEVERAGE

PROCESSING

1. Introduction

Lately, consumers have shown increased demand for preservative-free food of high quality with

fresh-like attributes. Consumer lifestyles have shifted towards increased consumption of raw and

unprocessed foods, while at the same time, the consumption of raw food has been linked to

outbreaks of food-borne illnesses. The shift in consumer demand toward more natural foods is

likely linked to the negative attributes which result from thermal processing, including the loss of

heat-sensitive nutrients and fresh-like sensory attributes.

Preservation of food extends its shelf life, largely through inactivation of the

microorganisms which contribute to spoilage. This end has been traditionally achieved by

processing foods at high temperatures or by adding chemical preservatives, but these methods have

many limitations. Food scientists and engineers have begun focusing on the use of alternative

processing technologies, where their main goals are the inactivation of pathogens and

microorganisms which contribute to spoilage while preserving fresh-like qualities. High pressure

processing, pulsed electric fields, and ultrasound are some promising alternatives to traditional

processing methods, and this chapter reviews the engineering aspect of these novel technologies.

It focuses on examining their advantages and limitations, discussing equipment design, energy

requirements during processing, and the challenges involved in adopting these processes on an

industrial scale.

55

2. High pressure processing

High pressure processing (HPP), also known as high hydrostatic pressure (HHP) processing, is a

mature nonthermal technology that has been recently commercialized in food processing

industries, mainly for the pasteurization of fruit and vegetable juices, guacamole, smoothies, meat,

etc. In this process, pre-packaged food is subjected to very high pressures, commonly in the ranges

of 50 to 900 MPa, in the presence of liquid media as a pressure transmitting fluid. The use of high

pressure in food preservation is not new; its efficacy was discovered more than a century ago (Hite,

1899), but due to the complexity involved in the manufacture and operation of HPP equipment it

has not been widely adopted in the food processing industry. In 1990, the first HP processed food

product (fruit jam) was launched in the Japanese market (Thakur and Nelson, 1998).

The primary objectives of any food preservation technique are to inactivate pathogenic and

spoilage-causing microorganisms and extend shelf life of the food under consideration. The

conventional thermal process fulfilled these goals, but due to the development of cooked flavor

and loss of nutritional quality, alternative methods to process food have been explored. In high

pressure processing, only the secondary and tertiary structure of a protein is altered, which causes

the destruction of microorganisms with minimal or no impact on the quality attributes of the

processed food (Hayashi, 1989). This feature of HPP makes it as a sound alternative to traditional

processing methods.

2.1 HPP Equipment

The HPP system typically consists of a pressure vessel, a pump, two end closures, devices for

restraining the end closures (yoke, thread, and pin), and a control unit. Pressure vessels are

56

generally made of low-alloy steel of high tensile strength (Balasubramaniam et al., 2016).

Commercial scale vessels have a capacity in the range of 35 to 525 L whereas, pilot scale vessels

have capacities of 1 to 10 L. Pressure is applied using the pump, and can be achieved by direct or

indirect pressurization. In industrial setting, indirect pressurization is very common where an

electro-hydraulic pump is used to raise the pressure (Balasubramaniam et al., 2016).

HPP can run in either a batch or semi-continuous mode. During batch processing, pre-

packaged products are loaded into the pressure vessels. After the lid is closed, pressure is applied

to a pressure medium, usually a water/oil mix using a pump. During processing, heat is generated

by compression, resulting in a temperature rise of about 3°C per 100 MPa. After holding the

desired pressure for a given period, the pressure valve is released and the product discharged

(Balasubramaniam, Farkas, and Turek, 2008). Semi-continuous HPP is primarily performed before

product packaging, and is used for liquid products. In these systems, the liquid product is pumped

to pressure vessels by a transport pump and the supply valve is closed. The product inside the

vessels is pressurized by a high-pressure pump (Figure 5), and after the completion of the process,

the product is transferred to a filling system (Lelieveld and Hoogland, 2016).

57

Figure 5. Operation of HPP (Source: http://www.hiperbaric.com/).

The design of HPP to process food is based on the same parameters used in non-food

industries such as ceramics and chemicals (Balda, Aparicio, and Samson, 2012). There are two

types of design: vertical loading and horizontal loading. Horizontal product-loading systems are a

modified form of the vertical system that was originally adapted from non-food industries. GEL

ALSTOM ACB manufactured the first commercial horizontal-loading HPP system in 1998 (Jung,

Samson, and de Lamballerie, 2010).

Figure 6. Hiperbaric HPP system Hiperbaric-135 (Source: http://www.hiperbaric.com/).

58

Figure 7. Avure’s AV-30 HPP unit (Source: http://www.avure-hpp-foods.com/).

2.2 Compression heating and its effect

HPP is an adiabatic process which means the system is thermally isolated, and heat is generated

within the vessel by compression. During compression, the molecules are compressed and the

intermolecular forces between molecules release energy in the form of heat (Khurana, 2008).

The compression of any compressible substance results in generation of heat. Under

adiabatic conditions, fats and oils possess some of the highest compression heating values,

typically in the range of 6 – 8.7°C for a pressure change of 100 MPa (Rastogi et al., 2007), while

water has the lowest, increasing by only 3°C per 100 MPa (Koutchma, 2012). The fluid is used as

the medium for transmitting pressure, and the temperature of this pressure-transmitting fluid also

changes during adiabatic compression, causing changes in product temperature.

59

Assuming the process is truly adiabatic (i.e. the system possesses perfect thermal isolation),

the change in temperature resulting from pressurization can be derived:

Considering the change in entropy of the system (ds) that varies as a function of both temperature

(T) and pressure (P) of the system, i.e. S= f (T, P), then

𝑑𝑠 = (𝑑𝑠

𝑑𝑇) 𝑑𝑇 + (

𝑑𝑠

𝑑𝑃) 𝑑𝑃, (1)

If the process is assumed to be completely reversible, then the total change in entropy is

zero. By rearranging, the rate of heating due to compression (dT/dp) can be expressed as

𝑑𝑇

𝑑𝑝= −

𝛿𝑠

𝛿𝑃𝛿𝑠

𝛿𝑇

, (2)

𝑑𝑇

𝑑𝑝= −

𝑉(𝛿𝑠

𝑉𝛿𝑃)

1

𝑇(𝑇𝛿𝑠𝛿𝑇

)

, (3)

This can be expressed more simply (Eq. 4), as the rate of compression heating is directly

proportional to -T∙α, where α is an expansion coefficient (1/K), and is inversely proportional to the

specific heat capacity of the medium (Cp, J/kg∙K):

𝑑𝑇

𝑑𝑝= −

𝑇𝛼

𝜌𝐶𝑝, (4)

This simplified equation describes the phenomenon, and can be correlated with activation

energy and z-value.

Furthermore, pressure alone has also a significant effect on reaction rate. Consider the ideal

gas law,

60

𝑝𝑉 = 𝑛𝑅𝑇, (5)

where p is pressure, V is volume, n is number of moles, R is the gas constant, and T is temperature

Rearranging Equation 5 yields

𝑝 =𝑛𝑅𝑇

𝑣, (6)

Since R and n are constant, the pressure applied is directly proportional to the temperature

and volume; compression reduces the volume, and thus it follows that an increase in pressure

increases the temperature and concentration (n/v) of the reactant. Changes in temperature and

concentration directly affect reaction rates.

Microbial inactivation by the combination of high pressure and antimicrobials can be

considered in light of these relationships. The application of antimicrobials alone is less effective

than a combined application of high pressure and antimicrobials. This makes sense; the application

of high pressure brings microbial cells and antimicrobials together, increasing the effective

concentration of antimicrobials. Hence, the “reaction rate” increases as described by Equation 6,

and the population of microorganisms is reduced more rapidly.

2.3 Energy consumptions

Energy consumed during high pressure processing can be divided into two forms: energy utilized

in the initial pressurization, and second, energy present in the form of heat (compression heating)

leading to the increase in system temperature (Eq. 7):

𝐸𝑡𝑜𝑡𝑎𝑙 = 𝐸𝑝 + 𝐸𝑙, (7)

61

where 𝐸𝑡𝑜𝑡𝑎𝑙 is the total energy of the system, 𝐸𝑝 the energy used to raise the pressure, and 𝐸𝑡 is

the thermal energy.

Energy to raise the pressure

According to the first law of thermodynamics,

𝑑𝑈 = 𝛿𝑊 + 𝛿𝑄, (8)

where dU is the internal energy, and δW is the energy due to work (expressed as Ep in the case of

HPP), and δQ is heat transfer between the system and its environment. In the case of HPP, the

vessel can be considered hermetic, so the system is under adiabatic conditions (no heat transfer

between the system and its environment). Hence, (2) becomes

𝑑𝑈 = 𝛿𝑊, (9)

Following the definition of work due to the application of pressure, (the pressure

transmitting fluid in the vessel), we can write the following equation:

𝛿𝑊 = −𝑃𝑑𝑉, (10)

where P is pressure in the vessel and dV is the change in volume. We need to know the

compressibility of the pressure transmitting fluid used. Volume is a function of pressure and

temperature; thus, we can define dV as in equation (5):

𝑑𝑉 = (𝛿𝑉

𝛿𝑃)𝑇𝑑𝑃 + (

𝛿𝑉

𝛿𝑇)𝑝𝑑𝑇, (11)

We can introduce the compressibility, β (Pa-1), at constant temperature:

62

𝛽 = −1

𝑉∗ (

𝛿𝑉

𝛿𝑃)

𝑇 , (12)

From Eq. (10), (11), and (12) we get,

𝑊 =1

2∗ 𝑉 ∗ 𝛽 ∗ (𝑃𝑓𝑖𝑛𝑎𝑙

2 − 𝑃𝑖𝑛𝑖𝑡𝑖𝑎𝑙2), (13)

Thermal energy due to compression

As previously discussed (Section 2.2), the system undergoes adiabatic heating due to

pressurization. Barbosa-Cánovas and Rodriguez (2005) estimated that thermal energy, Et, during

an adiabatic and reversible change, is equivalent to the compression energy applied during this

process at constant temperature:

𝜌 ∗ 𝐶𝑝 ∗𝛿𝑇

𝛿𝑡= 𝑇 ∗ 𝛼𝑝 ∗

𝛿𝑃

𝛿𝑡 , (14)

As a result, the total energy in the system can be calculated during the come-up time

(compression), the holding time, and the decompression.

2.4 Packaging requirements

Flexible polymeric packages are eminently suitable for high pressure processing. The packaging

of products to be subjected to HPP should possess the strength and flexibility necessary to survive

the large pressure changes involved with no loss of original size, shape, or function. Tolerance of

a ~15% change in volume is typically considered the threshold for suitable packaging. For this

reason, rigid containers such as glass bottles or metal tins are incompatible with HPP.

63

Commonly used packaging materials include polypropylene, polyethylene, and nylon-

polypropylene pouches; flexible polymeric packages work well with HPP. As the package must

withstand very high pressures, and sometimes elevated temperatures, packages used for HPP

should have excellent mechanical properties as well as gas barrier properties. Aluminum-based

multiplayer polymeric packages tend to have a high degree of mechanical strength, but aluminum

has issues with blistering at high pressure (Han et al., 2006). Nylon-based polypropylene is another

suitable package compatible with HPP. Beverages are often packaged in PET bottles, which are

also compatible with HPP.

2.5 Factors affecting the process

2.5.1 Processing factors

a. Temperature

Temperature plays a very important role in high pressure processing as microbial inactivation

depends on the temperature. The initial temperature of the products to be treated and temperature

of the treatment chamber are two critical factors. Both of these temperatures have a strong

influence on microbial inactivation, as well any deterioration in food quality. Microbial

inactivation has shown to be enhanced by either increasing the initial temperature of food above

room temperature or by decreasing it (Kalchayanand et al., 1998). Temperatures above 70°C are

generally required to achieve commercial sterilization. HPP can also be used at refrigeration

temperature; this could be done to inactivate non-psychotropic cells (Patterson et al., 1995).

Temperature below -20°C have also shown good results for microbial reduction. At lower

temperatures, ice crystal formation can also play a role in microbial inactivation.

64

b. Pressure holding time

The pressure holding time is a period after come up time and before decompression; during this

period the pressure is held constant (Nguyen and Balasubramaniam, 2011). This time is crucial in

processing as it has a significant effect both on microbial inactivation as well as quality

deterioration; the shortest time capable of achieving microbial inactivation is desirable, as a

reduction in pressure holding time will increase throughput.

c. Compression heating

As discussed earlier, the physical compression experienced due to HPP leads to higher

temperatures of both the pressure-transmitting medium and the product being treated. The extent

of this increase in temperature depends on the type of the products, the initial temperature of the

product, and the pressure applied. It is also affected by pressure-transmitting medium,

pressurization type, vessel design, and rate of pressurization (Patazca et al., 2007).

Adiabatic heating in HPP may increase the temperature of food from 3 to 9°C/100 MPa

(Table 8). As soon as the pressure returns to its original level, the system returns to its original

temperature almost instantly. Foods with high fat content tend to experience a higher rise in

temperature compared to foods with high water content; Rasanayagam et al. (2003) observed that

the oil has little or no effect on its initial temperature during adiabatic heating.

65

Table 8. Adiabatic increments in temperature during HPP (de Heij et al., 2003).

Food products Initial temperature (°C) Temperature change (°C/100 MPa)

Water

20 2.8

60 3.8

80 4.4

Chicken 20 2.9

Milk fat 20 8.5

Figure 8. Time–temperature and pressure profiles of water at an initial temperature of 24.2ºC and set

pressure of 300 MPa during HPP (Source: Patazca et al., 2007).

66

2.5.2 Product factors

a. pH

Acidic food is ideal for processing by HPP; at lower pH, most microorganisms become more

sensitive to pressure treatment. During pressure treatment, there is a chance of ionization leading

to a decrease in pH (Hoover et al., 1989); for example, a decrease of 0.2 units in the pH of apple

juice with an increase in pressure by 100 MPa has been reported (Hermans, 1995). An increase in

acidity alone is typically not enough to reduce microbial loads to desired levels, but combining

HPP with a decrease in pH can massively increase its efficacy, exhibiting a synergistic rather than

an additive improvement. Ritz et al. (2008) observed no significant effect on the reduction (<1 log)

of L. monocytogenes in buffer at pH 7 processing at 4°C, but when pH was changed to 4.5, there

was a significant reduction (8.9 log) in the microbial population.

b. Water activity (aw)

Water activity plays a critical role in the inactivation of microorganisms by high pressure. A low

water activity level in food protects microorganisms from inactivation by HPP. Hayman et al.

(2008) stated that low water activity results in protein stabilization, protecting the microorganisms

from being inactivated by HPP. Pressure-injured microorganisms are more sensitive to low water

activity than regular or un-injured microorganisms.

There are many constituents in food that can change the water activity and prevent

inactivation by HPP; in general carbohydrates are found to be more protective than salts. However,

Patterson et al. (1995) found that the addition of 2% salt significantly reduced the recovery of some

pressure-treated microbes. Reducing the concentration of peptone water below 60% (aw = 0.8) has

67

been shown to significantly increase the susceptibility of L. monocytogenes to HPP (Hayman et

al., 2008). Oxen and Knorr (1993) reported 7 log reductions of Rhodotorula rubra after HPP

treatment (400 MPa, 25°C, 15 min) when water activity was greater than 0.96, but when it was

reduced below 0.91, no inactivation was observed. Similarly, Zygosaccharomyces bailii was

observed to completely inactivate by HPP at aw values greater than 0.8, but HPP treatment at lower

aw resulted in a greater population of survivors (Palou et al. 1997). The same authors concluded

that added sugar acted as a baroprotective agent, preventing inactivation of target microorganisms

by HPP.

The baroprotective effect depends on the type of solute added. Setikaite et al. (2009)

compared the effects of some additives, including sugars and salt, on HPP treatment to eradicate

E. coli K12. In their study, salt was the best choice for increasing the effects of HPP, glycerol and

fructose were somewhat less effective, and sorbitol was the most baroprotective additive.

c. Food composition

The composition of food also plays a vital role in the effectiveness of high pressure processing.

Many food constituents can protect microorganisms from inactivation by HPP (Hoover et al.,

1989). Large amounts of sugar, fat, and protein can act as baroprotectants, contributing to

microorganism resistance towards HPP.

68

2.6 Advantages and limitations of HPP

Advantages

An environmentally friendly process, requires only electricity and water (which is

recycled).

Preserves the freshness, sensory properties and nutritional quality of products.

Can easily destroy pathogens like Listeria, Salmonella, Vibrio, and Norovirus.

Can help develop clean label products.

Can also be used in product modification such as starch gelatinization, gel formation, and

meat tenderization.

Commercial sterilization of food is possible using HPP (>600 MPa) at 70-90°C.

Limitations

Can only be used with cut, blended, and clean fruits or vegetables; it does not work well

with whole and raw products.

Can change the texture of food products due to cell wall rupture.

Does not work with dry products due to their low water content; requires the presence of

water to inactivate microorganisms.

Some food enzymes are resistant to pressure.

69

Degradation of food can occur even after processing due to residual oxygen and enzyme

activity.

3. Pulsed electric fields

Processing juices and other beverages with pulsed electric fields (PEF) involves the application of

short pulses of high electric fields intensity (10-50 kV/cm) to products as they move between two

electrodes for a duration of microseconds to milliseconds (Pataro et al., 2011). PEF has shown

very promising results on the inactivation of vegetative microorganisms and spores when

combined with microbiological stress factors such as temperature changes, pH reduction, and

antimicrobial agents.

The inactivation of microorganisms by PEF is mainly due to electroporation of cell

membranes. PEF causes the breakdown of microorganism cell membranes by creating new pores

or by expanding existing ones (Ramaswamy et al., 2005). This results in leakage of their

cytoplasmic content, causing microbial death. The size and number of pores formed due to

electroporation depends on various factors such as electric field intensity, number of pulses, pulsed

width, processing temperature, and treatment time. Inactivation of microorganisms by PEF also

depends on product factors such as ionic strength or electrical conductivity of the food.

In PEF processing, electric field generates a surface charge on cell membranes; because of

the difference in charge state between a cell’s inner and outer layers, the cell membrane is

compressed, destabilizing the lipid layers and proteins of which it is composed. This compression

results in a thinner, more permeable membrane, which allows small molecules to enter the cell,

70

and eventually results in cell lysis due to membrane rupture. Higher field intensity increases the

amount of accumulated surface charge, and by extension, increases the rate of cell lysis.

Figure 9. Electroporation in microbial cell after PEF treatment.

3.1 PEF equipment

PEF treatment systems require a high voltage power supply. During processing, the liquid food

passes through a temperature-regulated treatment chamber centered between electrodes (high

voltage and ground), and insulated from the rest of the system. The electrodes, typically made of

stainless steel, but sometimes of other metals, pass current through food in the treatment chamber.

The temperature of the food product (which may increase upon exposure to electric fields) is

monitored by thermocouples, and a cooling or heating bath regulates its temperature, chilling or

heating to a set temperature, or maintaining room temperature, as required. After passing through

the treatment chamber, the processed food is pumped to an aseptic packaging unit.

71

Figure 10. Schematic diagram of a pulsed electric fields system

a

b

Figure 11. Pilot scale PEF systems: a. ELEA technologies (15 kW) and b. Diversified technologies Inc.

(DTI) (25 kW) (Source: http://elea-technology.de/ and http://www.divtecs.com/)

72

3.1.1 Electronic components of PEF systems

The electrical components of PEF systems consist of a pulse generator, capacitor, and a discharge

switch. The design of the discharge switch and circuit determine the pulse shape. The pulse

generator controls the length and voltage of the pulses applied to the system via a pulse forming

network (PFN): first, a capacitor is charged to a target voltage by the DC power supply. This

converts AC power of 50-60 Hz to high voltage AC, then finally converts it to the higher voltage

DC power required for PEF (Zhang et al., 1995). Then, the capacitors rapidly pulse the stored

energy though the food, a process controlled by an electric discharge switch. The energy input is

regulated by charging resistors; the amount of energy necessary to build the initial charge varies

based on the capacity and number of capacitors; more capacitors will increase the energy storage

capacity of the system. The amount of energy (E) stored in the capacitor can be determined using

Eq. 16.

𝐸 =1

2. 𝐶. 𝑉2, (16)

where C is the capacitance and V is the voltage.

Figure 12. Pulse forming network (PFN) in PEF system

73

3.1.2 Treatment chamber

The treatment chamber is a particularly important component for PEF treatment, as it is the site at

which the electric field is applied to the food to be treated. It consists of electrodes and insulators;

where multiple electrodes could be connected in series. Since the electrodes in the chamber come

into direct contact with the food being processed, food-grade materials must be used. Stainless

steel is the widely used to construct electrodes (Zhang et al., 1995; Angersbach et al., 2000). Other

materials such as gold, platinum or carbon can also be used to make electrodes (Bushnell, 1993).

Insulator can be made up of any non-conductive and heat resistant materials such as nylon,

polysulfone, polythene (Sale and Hamilton, 1967; Dunn and Pearlman, 1987; Zhang et al., 1995).

Food may be held in a static chamber or pumped through a continuous chamber. While a

static chamber is useful in small-scale applications, such as initial studies, a continuous chamber

is more appropriate for operation on an industrial scale. In either case, an oscilloscope hooked up

to the treatment chamber monitors the voltage and current passing through the chamber, and the

total electric field strength applied. As liquid food is passed through treatment chamber, it is

subjected to a pulse of electricity of set duration. The flow of fluid is adjusted by the pump, and

choice of the appropriate rate depends upon the conductivity of the liquid, its viscosity, and

diameter of the pipe.

74

Figure 13. PEF treatment chamber

3.2 Electric fields generation

According to Faraday’s law, a positive charge (q) at a given point within the treatment area

generates an electric field (Ef) with force (F). The strength of the electric field per unit charge can

thus be expressed as in Eq 17:

𝐸𝑓 =𝐹

𝑞 (17)

Different pulse shapes (e.g. square, bipolar, oscillatory, exponential decay) can be

generated depending on the system parameters. Regardless of pulse shape, generation of a pulsed

field requires rapid energy discharge to create extremely short pulse widths. Charging, on the other

hand, can be slow, as the recovery time is long relative to the pulse width. The desired electric

75

field intensity (Ef) is directly proportional to the charging voltage (V), and inversely proportional

to the distance (d) between the electrodes and it can be calculated for two parallel plate electrodes:

𝐸𝑓 =𝑉

𝑑 (18)

Pulse width (pulse duration) is the distance between two peaks of the waves, and is

controlled by the frequency applied. PEF treatment time can be calculated from the pulse width

and the number of pulses.

An increase in the voltage applied will increase the intensity of the applied electric field,

so, in general, higher voltage, leads to a faster reaction rate. The power input during pulsed electric

field processing can be calculated according to the following relationship,

𝑃 (𝑘𝑊) = 𝑉. 𝐼. 𝑥. 𝑓, (19)

where V is the voltage in kW, I is applied current, x is the pulse width in μs, and f is the frequency

in MHz. Thus, increases in any of the terms, voltage, pulse width or frequency, will increase the

power input, which in turn will generate heat and increase the temperature of the liquid in the

treatment chamber. The electrical conductivity of the fluid increases as the temperature rises; over

time, this may cause excess current flow, leading to system shutdown.

3.3 Energy consumption

The specific energy input (W) for each pulse can be expressed in kJ/kg, calculated according to

Eq. 20:

𝑊 =1

𝑚. 𝑉. 𝐼. 𝑡, (20)

76

where m is the mass of food contained within the treatment chamber in kg, V is the input voltage

in kV, I is the current intensity in amp, and t is the treatment duration in μs. Total specific energy

can be calculated by multiplying W by the number of pulses.



a. Electric field intensity

The applied electric field intensity plays a largest role on the inactivation of microorganisms by

PEF (Hülsheger & Niemann, 1980). The effects of PEF on microbial inactivation have been shown

to depend mainly on electric field intensity and the duration of exposure to the electric field; other

factors do not play significant roles (Sale and Hamilton, 1967). A critical electric field intensity

(Ec) threshold must be reached for PEF to be effective (Hülsheger et al., 1981). The Ec needed for

the inactivation of microorganisms depends on the cell size, its orientation, and transmembrane

potential of cells (Jeyamkondan et al., 1999; Heinz et al., 2002). It is worth mentioning that

transmembrane potentials is larger for larger cells. Hence, smaller cells require higher field

intensity then larger cells. Toepfl et al. (2005), found that inactivation of Listeria innocua (small

cell) requires a minimum of 15 kV/cm whereas, S. cerevisiae (large cell) can be inactivated with

the field intensity of around 2-4 kV/cm.

b. Treatment time

Total treatment time is calculated by multiplying pulse width and the number of pulses. Choice of

appropriate pulse width is vital for effective treatment. While intuitively, longer pulses may sound

77

desirable, they can lead to electrodeposition on the surface of the electrodes and shorter pulses

should be used (Zhang et al., 1994). Neumann et al. (1992) found that pulses with a duration of 1-

5 µs caused the highest levels of microbial inactivation; they hypothesized that a period of only 10

ns is required to establish the required difference in potential across the cell membrane to initiate

electroporation. The consensus is that the electric field must be maintained above the critical

threshold for an additional 1-5 µs beyond the start of electroporation in order to damage the cell

membrane beyond the ability of microbes to repair (Schoenbach et al., 2000).

The length of treatment required for microbial deactivation (critical treatment time)

depends not only on pulse width, but also on the intensity of the electric field. As long as the field

intensity is greater than Ec, the critical treatment time decreases with increase in electric field

intensity up to a threshold of 1.5 x Ec, after which it remains constant (Barbosa-Cánovas et al.,

1999).

c. Pulse shape

While a wide variety of wave shapes are possible, only exponential decay and square-shaped

pulses are widely used in PEF. Among two types, square-shaped has shown to have more lethal

on microbial inactivation. Barbosa-Cánovas et al. (1999), reported that in square pulse, peak

voltage is maintained for longer period of time hence they are energy efficient and has better lethal

effect as compared to exponentially decaying pulses. In addition to lethal effect, square pulse is

also found to cause less electrolysis of food which cause less deposit on the electrode surface

(Zhang et al., 1995; Barbosa-Cánovas et al., 1999). There are two modes by which PEF can

generate pulses: monopolar and bipolar. Bipolar pulses have been found to be to be more effective

than monopolar pulses in microbial inactivation regardless of pulse shape (Qin et al., 1994). On

78

the other hand, few studies show no difference between two types of pulse on the inactivation of

both gram-positive and gram-negative bacteria (Beveridge et al., 2002, Evrendilek and Zhang,

2005).


Properties of a food product like water activity, pH, temperature, conductivity, and the presence

of air bubbles can all influence PEF microbial inactivation.

a. Conductivity/ Dielectric strength of food:

Conductivity is one of the important parameters that needs to be considered when planning to use

PEF. Foods with a higher ion content blocks transmission of the electric field across the chamber,

preventing supercritical field strength from building. In other words, when electrical conductivity

is high, generated electric fields have small peaks which has less killing effect (Barbosa-Cánovas

et al., 1999). Hence it can be said that juice and beverages with low electrical conductivities allows

the use of higher electric field which can results in higher inactivation (Alkhafaji and Farid, 2007).

This problem could be overcome by selecting the proper configuration of electrodes to achieve

higher resistivity.

b. Presence of air bubbles:

The presence of air bubbles changes the dielectric properties of the product resulting into dielectric

breakdown during PEF processing. It may also result into non-uniform treatment as well as arcing.

For this reason, sparkling beverages and foam forming products are not very suitable for PEF, in

79

case of sparkling beverages, PEF treatment prior to carbonation can be done. Vacuum degassing

or pressurizing is required for some product to remove trapped air.

c. Product temperature

As in other technologies, rise of temperature during processing is associated with PEF. The rise of

temperature has some positive impacts as moderate heat and PEF has shown synergism on the

inactivation of microorganisms. But some time while processing milk based beverages products,

the rise of temperature might cause coagulation of protein and deposit formation on the electrodes.

Hence it is necessary to control the temperature during processing; control can be done by placing

the chiller in between treatment chambers connected in series.

The inlet product temperature is raised using an external source of heat by using a heat

exchanger. Then there is addition of certain amount of heat during processing, and finally the

treated product is cooled down immediately to prevent from thermal damage of nutrients. PEF act

either additively or synergistically with mild-heat in microbial inactivation. Additive effect of PEF

and mild-temperature (50-60°C) has been observed in many studies (Rowan et al., 2001; Craven

et al., 2008). Synergist effect between PEF and temperature (35-65°C) was observed by Heinz et

al. (2003), who also found the reduction of energy consumption from 100 kJ/kg to 40 kJ/kg to

achieve 6 log reduction of E. coli in apple juice when PEF was combined with heat. Synergistic

effect of PEF and heat is due to change in fluidity of the cell membrane at higher temperature

which cause the cell more susceptible to electroporation by PEF (Jayaram et al., 1992).

80

3.5 Advantages and limitations of PEF

Advantages

Takes only a short time to inactivate vegetative microorganisms including yeasts, spoilage

and pathogenic bacteria.

Capable of pasteurizing liquid foods and juices without addition of chemical preservatives.

Can be used for conventional thermal pasteurization of acidic or acidified food;

pasteurization of low-acid foods requires incorporation of other hurdles.

Pore formation in fruit and vegetable cells can increase extraction of juice and desirable

bioactive compounds from the tissues.

Can serve as an alternative to enzymatic maceration and mechanical disintegration.

Limitation

Runs in a continuous mode and must done before packaging, requiring the use of costly

aseptic filling techniques.

Initial investment is high; an entry-level industrial-scale pulser may cost in the

neighborhood of $250,000, while others may cost $450,000 to $2,000,000.

Only proven to be useful to inactivate vegetative microorganisms. Some spores and

enzymes have shown a high degree of resistant towards PEF, though incorporation of other

hurdles can solve these issues.

81

A product must be pumpable, possess low electrical conductivity and no air bubbles. The

presence of particles is also a limiting factor; particle size should be smaller than the

internal diameter of the electrode.

4. Ultrasound

Ultrasound (US) is a novel technology that has shown promising results in the preservation of fruit

and vegetable juices. It has been widely used in the food and bioprocess industry for many

purposes including extraction, homogenization, degassing, and antifoaming. Recent studies shows

that this technology can meet the US FDA’s requirement for a 5 log reduction of most resistant

and pathogenic microorganisms present in food products (Salleh-Mack and Roberts, 2007).

Figure 14. Schematic diagram of ultrasound processing

Ultrasound operation can be divided into two categories: low-intensity and high-intensity.

Low-intensity ultrasound uses low amplitude at high frequency (>1 MHz), and is commonly used

82

in structural analysis. This kind of ultrasound does not affect the physical structure. On the other

hand, high-intensity ultrasound, also called power ultrasound, uses high amplitude (typically >100

µm) at low frequency (20-100 kHz) (Kentish, 2014).

When an ultrasound wave produced by the transducer is propagated into the liquid medium

through the sonotrode, there is a mechanical generation of pressure, which in turns releases heat.

When this ultrasound wave passes into liquid with a different pressure gradient, the formation of

bubbles takes place. These bubbles form and collapse thousands of times per second; this rapid

formation and collapse of bubbles is called cavitation, and is a key factor in ultrasound processing.

Two types of bubbles can be formed during processing: stable and transient bubbles. Stable

bubbles are very small, retain their size, and don’t collapse, whereas transient bubble increase in

size during processing and break when it reaches its critical value. During the breaking of those

transient bubbles, a large amount of heat is generated (~5000 K) at high pressure (>1000 atm)

(Feng et al., 2011). These bubbles act as microreactors where cell membranes can rupture.

83

Figure 15. Creation of stable cavitation bubbles; and creation and the collapse of transient cavitation

bubbles. (a) displacement; (b) transient cavitation; (c) stable cavitation; (d) pressure (Source: Santos et al.,

2009).

Bubble size depends upon the frequency, amplitude, pressure, and temperature of the liquid

medium. Leighton (1994) developed an empirical relation to estimate the size of bubbles; this

relation can be given by:

𝑓. 𝑟 = 3, (21)

where f is the frequency in MHz, and r is the bubble radius in μm.

4.1 Ultrasound equipment

Ultrasound equipment consists of a wave generator, transducer, and treatment chamber. The

transducer is responsible for the production of electrical or mechanical energy and its conversion

into sound energy in the ultrasonic frequency range (Ercan and Sousal, 2013).

84

Figure 16. Ultrasound equipment: Lab scale (Left) and Industrial scale (Right) (Source:

www.hielscher.com)

High frequency ultrasonic waves are produced via the piezoelectric effect. The most

common method is pulsed excitation of a ceramic crystal contained within a transducer. There are

two main transducer types in common use.

Magnetostrictive transducers produce ultrasonic waves via application of electric pulses to

a nickel core, with coils wrapped around opposing sides. They are capable of producing ultrasonic

frequences below 100 kHz with about 60% efficiency. Piezoelectric transducers are far more

energy efficient (~95%), and thus are the type commonly used in ultrasonic cleaners. They consist

of a transducer sandwiched between metal blocks, where it is more protected than the crystal in a

magnetostrictive transducer.

85

4.2 Propagation and attenuation of ultrasonic waves

The propagation of ultrasonic wave in liquid medium depends on applied acoustic pressure and

this can be represented on the basis of sinusoidal wave’s theory (Patist and Bates, 2008). In a

sinusoidal wave, acoustic pressure depends on frequency, time, and maximum pressure amplitude

of the wave (Ashokkumar, 2006). This relation can be given by,

𝑃𝑎 = 𝑃𝑎,𝑚𝑎𝑥 sin(2𝜋𝑓𝑡), (25)

where f is the frequency of the applied ultrasound wave, t is the time, and 𝑃𝑎,𝑚𝑎𝑥 is the maximum

pressure amplitude of the wave. Equation 25 can be explained on the basis of the intensity of the

wave, i.e., applied amplitude. When low amplitude is used, the pressure wave creates a motion

which mixes within the fluid, a phenomenon called acoustic streaming (Patist and Bates, 2010).

At high amplitude, the formation of small bubbles at existing nuclei in the fluid take places (Patist

and Bates, 2011). The negative transient pressure in the liquid support the growth of bubbles and

produces new cavities due to cushioning effects. The bubble grows over a number of cycles and

finally collapse (Mason, 1998).

Wave intensities decay due to adsorption of ultrasound waves by particles, wave scattering,

and mode conversion. The attenuation of ultrasound waves corresponds to decay in wave

amplitude. The amplitude decay can be calculated theoretically using Arrhenius equations (Jensen

et al., 2011):

𝐴(𝑧) = 𝐴0𝑒−𝜇𝐴𝑧 , (27)

where 𝜇𝐴is the amplitude attenuation factor.

86

The attenuation coefficient (𝛼) can be defined as:

𝛼 = 20 log(𝑒). 𝜇𝐴 =≅ 8.7𝜇𝐴 , (28)

[20 log Az/A0 is the amplitude drop in decibels]

The adsorption coefficient of the particles depends on the applied frequency. It is given by:

𝑎 = 𝛼/𝑓𝑏 (b ~ 1), (29)

Putting the value of 𝜇𝐴 in equation (8), we get:

𝐴(𝑧, 𝑓) = 𝐴0𝑒−𝑎𝑓𝑧/8.7, (30)

The decay wave intensity in ultrasound can be quantified using Eq. 30.

4.3 Energy consumption

During processing, the transducer converts electrical energy into sound energy, which vibrates at

a specific frequency. The amount of energy released from the ultrasound depends on the applied

frequency and amplitude, as given by (Kentish and Feng, 2014):

𝐸𝑛𝑒𝑟𝑔𝑦 (𝐸) = 𝑘𝑓2𝐴2, (31)

where k is the dissipation constant, f is the frequency, and A is the amplitude.

Energy consumption during processing can be calculated using time and temperature data,

specifically the temperature increase during processing. The calorimetric method can be used to

quantify the work done (Eq. 32),

87

𝑊𝑜𝑟𝑘 𝑑𝑜𝑛𝑒 (𝑄) = 𝑚. 𝐶𝑝. 𝑑𝑇/𝑑𝑡 , (32)

where m is the mass of juice (kg), Cp is the specific heat of product, and dT/dt is the change in

temperature with respect to time.

Acoustic energy density (AED) can be calculated by dividing the total work done (Q) by

the volume of product (V, mL) (Eq. 33) (Tiwari and Mason, 2012).

𝐴𝐸𝐷 =𝑄

𝑉 , (33)

Ultrasonic intensity can be calculated by dividing the work done by area of the sonotrode

(A, cm2) (Eq. 34).

𝑈𝐼 =𝑄

𝐴 , (34)



Microbial inactivation by ultrasound depends on applied amplitude and frequency. Frequencies of

20 to 60 kHz and amplitudes of 60 to 120 µm are used in food processing. High amplitude is more

efficient in the inactivation of microorganisms than to low amplitude (Wordon et al., 2011).

a. Frequency of sound waves

Application of high frequency generates smaller cavitation bubbles (Feng et al., 2011). At higher

frequency, wavelength is shorter, so bubbles have less time to travel before they collapse hence

the bubbles are smaller. On the other hand, at a low frequency, the wavelength is longer, the

88

bubbles get more time to travel along the wave before collapsing, so the size of the bubbles is

bigger. Bubble formation does not occur at frequencies above 2.5 MHz (Alliger, 1975).

Figure 17. Effect of frequency on the size of bubbles. (Source: http://www.ctgclean.com/).

b. External pressure

Large amounts of energy are released when bubbles collapse in the presence of pressure, as the

bubble collapses more rapidly and violently (Lorimer and Mason, 1987).

c. Intensity

The intensity in the ultrasound treatment is directly related to the amplitude of the waves. A certain

threshold intensity is required to start the cavitation. High amplitude is always not desired in

extraction of intracellular components from tissues, but is desirable for microbial inactivation. The

use of high amplitudes in ultrasound processing may have many disadvantages including

deterioration of the transducer, in addition it may results in agitation of liquid instead of cavitation

(Santos et al., 2009).

89


a. pH

pH of the liquid medium is a crucial factor in microbial inactivation. In general, lower medium pH

increases the rate of inactivation. Salleh-Mack and Roberts (2007) studied the effects of ultrasound

at two different pH values, 2.5 and 4.0, and found significant differences in the inactivation rate of

E. coli (ATCC 25922TM).

b. Temperature of product

Ultrasound treatment is an exothermic process i.e., there is release of heat. The propagation of

sound waves in the liquid medium depends on the temperature of the medium. The speed of sound

waves in water is 1482 m/s at 20 °C. At 37°C, the speed increases by 2.5 m/s (Bilaniuk and Wong,

1993). If the temperature of the liquid is increased during ultrasound processing, ultrasonic waves

can propagate more quickly. However, increasing temperature also reduces the cavitation effect:

at high temperatures cavitation bubbles collapse less violently, as the vapor pressure is higher, so

a balance must be made.

Regarding microbial inactivation, when ultrasound is used at mild temperature, efficient

inactivation can be achieved by the combined effects of ultrasound and heat; a synergistic effect

in microbial load reduction is observed (Piyasena et al., 2003; Feng et al., 2008). Wordon et al.

(2012) found a significant decrease in D-value of S. cerevisiae with ultrasound processing at 60°C

(D-value 0.73 min) as compared to temperature alone (D-value 3.53 min) and ultrasound alone

(D-value 3.1 min). Lee et al. (2009) studied the inactivation of E. coli K12 using ultrasound

combined with heat and/or pressurization. They observed that the addition of other lethal

90

parameters (temperature and/or pressure) to ultrasound treatment significantly reduced the time

required to achieve 5 log reduction.

c. Viscosity

Media with high viscosity impedes the passage of ultrasonic waves; thus, the propagation of

ultrasonic waves will be low, resulting in less cavitation effects and lower rates of microbial

inactivation. Liquid with high viscosity requires higher amplitudes, as viscous liquids resist the

flow of ultrasonic waves.

d. Density/ Particles

Inactivation is efficient in liquids without particulates, but when dense concentrations of particles

are present, ultrasonic waves cannot pass easily through the medium, and there is less cavitation.

The density of the liquid medium reflects the mass content of the particles in the medium. Clear

liquid (such as water) has a density of approximately 1000 kg/m3, while orange juice has a density

of 1048 kg/m3 due to the particles present in it. The propagation of ultrasound wave involves the

movement of the particles present in the medium. When particles are present in significant

amounts, more force is required to move the particles. This indicates that the higher the density,

the slower wave propagation will be. This can also be proved mathematically by using the Newton-

Laplace equation:

𝑐 = √𝐾

𝜌 , (31)

where c is the speed of the wave, K is the modulus of bulk elasticity, and 𝜌 is the density. Since K

is constant, wave speed is inversely proportional to the density of the medium.

91

Compressibility is the term that describes the compactness of the medium, similar to

density in the liquid medium. When the liquid has fewer particles, they are loosely compressed,

and ultrasonic waves can more easily pass through it.

4.5 Advantages and limitations of Ultrasound

Advantages

Very low energy consumption compared to thermal processes as well as other nonthermal

technologies.

Have multiple application in food processing: pasteurization, de-carbonation, emulsion

creation, homogenization, extraction of intracellular components.

No major degradation of flavor and bioactive compounds during processing.

Capital and operating costs are comparatively low.

Limitations

Heat can be released during processing, a process that is sometimes challenging to control.

The release of heat in the medium reduces the cavitation effect.

The volume of the product directly affects treatment efficacy, as acoustic energy density

depends on volume.

92

If the sonication probe is in direct contact with the food there is a chance of metal release

from sonotrode into the food. Appropriate food-safe materials should be selected for the

sonotrode.

Effects of ultrasound strongly depend on the position of the probe and size of treatment

vessels, so scale-up and reproducibility is challenging.

Most lab-scale ultrasound processing has been done in batch mode, although continuous

processing is possible. Extensive studies need to be done of the continuous mode to find

optimal conditions of amplitude and flow rate. When the liquid is in motion, ultrasonic

energy behaves differently than in batch mode, where the liquid is still. Residence time

also affects the results of ultrasound processing.

5. Final Remarks

This literature review presents a clear picture of what is known and the knowledge gap that need

to be filled in order to design a system for nonthermal processing of beverages. The basic

knowledge on design of equipment and the working mechanism is vital in preliminary stages of

process planning. By understanding the factors that affect the process, one can identify what kind

of products is suitable for a particular process and what precautions should be taken in choosing

the operating conditions. Every processing technologies has their own advantages and limitation;

optimization from cost and technical aspects is required in selecting the appropriate technology to

process a particular fruit and vegetable based juices and beverages.

93

References

Alkhafaji, S. R., & Farid, M. (2007). An investigation on pulsed electric fields technology using

new treatment chamber design. Innovative Food Science and Emerging


Alliger, H. (1975). Ultrasonic disruption. American Laboratory, 10(10), 75-85.

Angersbach, A., Heinz, V., & Knorr, D. (2000). Effects of pulsed electric fields on cell membranes

in real food systems. Innovative Food Science and Emerging Technologies, 1(2), 135-149.

Ashokkumar, M., Sunartio, D., Kentish, S., Mawson, R., Simons, L., Vilkhu, K., & Versteeg, C.

K. (2008). Modification of food ingredients by ultrasound to improve functionality: a

preliminary study on a model system. Innovative Food Science and Emerging


Balasubramaniam, V. M., Barbosa-Cánovas, G. V., & Lelieveld, H. L. (2016). High-Pressure

Processing Equipment for the Food Industry. In High Pressure Processing of Food (pp.

39-65). Springer New York.

Balasubramaniam, V. M., Farkas D., Turek, E. J. (2008). Preserving Foods through High-Pressure

Processing. Food Technology 11, 32-38.

Balasubramaniam, V. M., Ting, E. Y., Stewart, C. M., and Robbins, J. A. (2004). Recommended

laboratory practices for conducting high-pressure microbial inactivation experiments.

Innovative Food Science and Emerging Technologies, 5: 299-306.

94

Balda, F. P., Aparicio, B. V., & Samson, C. T. (2012). Industrial high pressure processing of foods:

review of evolution and emerging trends. Journal of Food Science and Engineering, 2(10),

543.

Barbosa-Cánovas, G. V., & Rodríguez, J. J. (2005). Thermodynamic aspects of high hydrostatic

pressure food processing. Novel Food Processing Technologies, 183-205.

Barbosa-Canovas, G. V., Pothakamury, U. R., Gongora-Nieto, M. M., & Swanson, B. G.

(1999). Preservation of Foods with Pulsed Electric Fields. Academic Press.

Bates, D., & Patist, A. (2010). Industrial applications of high power ultrasonics in the food,

beverage and wine industry. Case Studies in Novel Food Processing Technologies:

Innovations in Processing, Packaging, and Predictive Modelling, 119-138.

Bilaniuk, N., & Wong, G. S. (1993). Speed of sound in pure water as a function of temperature. The

Journal of the Acoustical Society of America, 93(3), 1609-1612.

Bushnell, A. H., Dunn, J. E., Clark, R. W., & Pearlman, J. S. (1993). U.S. Patent No. 5,235,905.

Washington, DC: U.S. Patent and Trademark Office.

de Heij, W. B., Van Schepdael, L. J. M. M., Moezelaar, R., Hoogland, H., Matser, A. M., & van

den Berg, R. W. (2003). High-pressure sterilization: Maximizing the benefits of adiabatic

heating. Food Technology, 57(3), 37-41.

Dunn, J. E., & Pearlman, J. S. (1987). U.S. Patent No. 4,695,472. Washington, DC: U.S. Patent

and Trademark Office.

Ercan, S. Ş., & Soysal, Ç. (2011). Effect of ultrasound and temperature on tomato

peroxidase. Ultrasonics Sonochemistry, 18(2), 689-695.

95

Feng H, Yang W, Hielscher T. (2008). Power ultrasound. Food Science and Technology

International, 14, 433-436.

Feng, H., Barbosa-Cánovas, G. V., & Weiss, J. (2011). Ultrasound Technologies for Food and

Bioprocessing. New York: Springer.

Han, J., & Yuan, J. (2007). Advances in Packaging for Nonthermal Processes.

Hayman, M. M., Kouassi, G. K., Anantheswaran, R. C., Floros, J. D., & Knabel, S. J. (2008).

Effect of water activity on inactivation of Listeria monocytogenes and lactate

dehydrogenase during high pressure processing. International Journal of Food


Hayman, M. M., Kouassi, G. K., Anantheswaran, R. C., Floros, J. D., & Knabel, S. J. (2008).

Effect of water activity on inactivation of Listeria monocytogenes and lactate

dehydrogenase during high pressure processing. International Journal of Food


Heinz, V., Alvarez, I., Angersbach, A., & Knorr, D. (2001). Preservation of liquid foods by high

intensity pulsed electric fields—basic concepts for process design. Trends in Food Science


Hoover, D. G., Metrick, C., Papineau, A. M., Farkas, D. F., & Knorr, D. (1989). Biological effects

of high hydrostatic pressure on food microorganisms. Food Technology (USA).

Hülsheger, H., & Niemann, E. G. (1980). Lethal effects of high-voltage pulses on E. coli

K12. Radiation and Environmental Biophysics, 18(4), 281-288.

96

Hülsheger, H., Potel, J., & Niemann, E. G. (1981). Killing of bacteria with electric pulses of high

field strength. Radiation and Environmental Biophysics, 20(1), 53-65.

Jayaram, S., Castle, G. S. P., & Margaritis, A. (1992). Kinetics of sterilization of Lactobacillus

brevis cells by the application of high voltage pulses. Biotechnology and

Bioengineering, 40(11), 1412-1420.

Jensen, F. B., Kuperman, W. A., Porter, M. B., & Schmidt, H. (2011). Wave propagation theory,

In Computational Ocean Acoustics. Springer Science & Business Media.

Jeyamkondan, S., Jayas, D. S., & Holley, R. A. (1999). Pulsed electric field processing of foods:

a review. Journal of Food Protection, 62(9), 1088-1096.

Jung, S., Samson, C. T., & de Lamballerie, M. (2010). High hydrostatic pressure food processing.

In Alternatives to Conventional Food Processing, 254-306.

Kalchayanand, N., Sikes, A., Dunne, C. P., & Ray, B. (1998). Factors influencing death and injury

of foodborne pathogens by hydrostatic pressure-pasteurization. Food Microbiology, 15(2),

207-214.

Kentish, S., & Feng, H. (2014). Applications of power ultrasound in food processing. Annual

Review of Food Science and Technology, 5, 263-284.

Koutchma, T. (2012). Design for High‐Pressure Processing. In Rahman, S., & Ahmed, J. (Eds.).

Handbook of Food Process Design, John Wiley & Sons.

Lee, H., Zhou, B., Feng, H., & Martin, S. E. (2009). Effect of pH on inactivation of Escherichia

coli K12 by sonication, manosonication, thermosonication, and

manothermosonication. Journal of Food Science, 74(4), 191-198.

97

Leighton, T. (2012). The acoustic bubble. Academic press.

Lelieveld, H. L., & Hoogland, H. (2016). Continuous High-Pressure Processing to Extend Product

Shelf Life. In High Pressure Processing of Food (pp. 67-72). Springer New York.

Lorimer, J. P., & Mason, T. J. (1987). Sonochemistry. Part 1—the physical aspects. Chemical

Society Reviews, 16, 239-274.

Mason, T. (1998). Power ultrasound in food processing—the way forward. In Povey M. J. W.,

Mason T. J. (Eds) Ultrasound in Food Processing, Glasgow: Blackie Academic &

professional, 104-124.

Nguyen, L.T., & Balasubramaniam, V. M. (2011). Fundamentals of food processing using high

pressure. Zhang, H. Q., Barbosa-Canovas, G.V., Balasubramaniam, V.M., Patrick Dunne,

C., Farkas, D.F., and Yuan, J.T.C. eds. Nonthermal Processing Technologies for Food. IFT

Press, Wiley-Blackwell, Chichester, West Sussex, UK, 3-19.

Oxen, P., & Knorr, D. (1993). Baroprotective effects of high solute concentrations against

inactivation of Rhodotorula rubra. Lebensmittel Wissenschaft Technologie, 26, 220–223.

Palou, E., Lopez-Malo, A., Barbosa-Canovas, G. V., Welti-Chanes, J., & Swanson, B. G. (1997).

Kinetic Analysis of Zygosaccharomyces bailii Inactivation by High Hydrostatic

Pressure. LWT-Food Science and Technology, 30(7), 703-708.

Pataro, G., Senatore, B., Donsì, G., & Ferrari, G. (2011). Effect of electric and flow parameters on

PEF treatment efficiency. Journal of Food Engineering, 105(1), 79-88.

98

Patazca, E., Koutchma, T., & Balasubramaniam, V. M. (2007). Quasi-adiabatic temperature

increase during high pressure processing of selected foods. Journal of Food

Engineering, 80(1), 199-205.

Patist, A., & Bates, D. (2008). Ultrasonic innovations in the food industry: From the laboratory to

commercial production. Innovative Food Science and Emerging Technologies, 9(2), 147-

154.

Patist, A., & Bates, D. (2011). Industrial applications of high power ultrasonics. In Feng, H.,

Barbosa-Cánovas, G. V., & Weiss, J. (Eds) Ultrasound Technologies for Food and

Bioprocessing (pp. 599-616). Springer New York.

Patterson, M. F., Quinn, M., Simpson, R., & Gilmour, A. (1995). Sensitivity of vegetative

pathogens to high hydrostatic pressure treatment in phosphate-buffered saline and

foods. Journal of Food Protection, 58(5), 524-529.



Qin, B. L., Zhang, Q., Barbosa-Canovas, G. V., Swanson, B. G., & Pedrow, P. D. (1994).

Inactivation of microorganisms by pulsed electric fields of different voltage

waveforms. IEEE Transactions on Dielectrics and Electrical Insulation, 1(6), 1047-1057.

Ramaswamy, R., Jin, T., Balasubramaniam, V. M., & Zhang, H. (2005). Pulsed electric field

processing: fact sheet for food processors. Ohio State University Extension Factsheet, 22.

99

Rasanayagam, V., Balasubramaniam, V. M., Ting, E., Sizer, C. E., Bush, C., & Anderson, C.

(2003). Compression heating of selected fatty food materials during high‐pressure

processing. Journal of Food Science, 68(1), 254-259.

Ritz, M., Jugiau, F., Federighi, M., Chapleau, N., & De Lamballerie, M. (2008). Effects of high

pressure, subzero temperature, and pH on survival of Listeria monocytogenes in buffer and

smoked salmon. Journal of food protection, 71(8), 1612-1618.

Sale, A. J. H., & Hamilton, W. A. (1967). Effects of high electric fields on microorganisms: I.

Killing of bacteria and yeasts. Biochimica et Biophysica Acta (BBA)-General

Subjects, 148(3), 781-788.

Salleh-Mack, S. Z., & Roberts, J. S. (2007). Ultrasound pasteurization: the effects of temperature,

soluble solids, organic acids and pH on the inactivation of Escherichia coli ATCC

25922. Ultrasonics Sonochemistry, 14(3), 323-329.

Santos, H. M., Lodeiro, C., and Capelo- Martínez, J. L 2009. The power of ultrasound. In Capelo-

Martínez, J. L. (Ed.). Ultrasound in Chemistry: Analytical Applications. John Wiley &

Sons.

Setikaite, I., Koutchma, T., Patazca, E., & Parisi, B. (2009). Effects of water activity in model

systems on high-pressure inactivation of Escherichia coli. Food and Bioprocess

Technology, 2(2), 213-221.

Thakur, B. R., & Nelson, P. E. (1998). High‐pressure processing and preservation of food. Food

Reviews International, 14(4), 427-447.

100

Tiwari, B. K., & Mason, T. J. (2012). Ultrasound processing of fluid foods. In Novel Thermal and

Non-Thermal Technologies for Fluid Foods (pp. 135-165).

Toepfl, S., Heinz, V. and Knorr, D. (2005) Overview of Pulsed Electric Field Processing for Food.

In Emerging Technologies for Food Processing. Ed. Da-Wen Sun. pp. 69-99. Elsevier

Academic Press, London.

Ugarte‐Romero, E., Feng, H., Martin, S. E., Cadwallader, K. R., & Robinson, S. J. (2006).

Inactivation of Escherichia coli with power ultrasound in apple cider. Journal of Food

Science, 71(2), E102-E108.



and heat for the application of hurdle technology. Food Research International, 47(2),

134-139.

Zhang, Q., Barbosa-Cánovas, G. V., & Swanson, B. G. (1995). Engineering aspects of pulsed

electric field pasteurization. Journal of Food Engineering, 25(2), 261-281.

101

CHAPTER THREE

COMBINED EFFECT OF ULTRASOUND AND MILD TEMPERATURES ON THE

INACTIVATION OF E. coli IN FRESH CARROT JUICE AND CHANGES

ON ITS PHYSICOCHEMICAL CHARACTERISTICS

Abstract

The combination of ultrasound and mild temperatures to process fruits and vegetables juices is a

novel approach that is showing promising results for microbial inactivation and preservation of

bioactive compounds and sensory attributes. This study centers on investigating the inactivation

of E. coli (ATCC 11755TM) in carrot juice as a result of the combined effect of ultrasound (24 kHz

frequency, 120 µm, and 400 W) with temperature (50, 54, and 58ºC) and processing time (0-10

min). In addition, the possible changes in physicochemical properties and the retention of bioactive

compounds after processing were analyzed. Microbial inactivation with ultrasound treatment at

50°C resulted in 3.5 log reduction after 10 min, whereas at 54°C almost 5 log reduction was

attained in the same period of time; meanwhile, for treatment at 58°C, no viable cells were detected

(>5 log reduction) after 2 min. There was no significant difference (p>0.05) on pH (6.80-6.82),

ºBrix (8.0-8.5), titratable acidity (0.29-0.30%), total carotenoid (1774-1835 µg/100 ml), phenolic

compounds (20.19-20.63 µg/ml), ascorbic acid (4.8 mg/100 ml), and color parameters between

fresh and ultrasound treated samples at the studied temperatures. To predict the inactivation

patterns, observed values were tested using three different general models: first-order, Weibull

distribution, and biphasic. The Weibull and biphasic models show good correlation for inactivation

under all processing conditions. Results show ultrasound in combination with mild temperature

102

could be effectively used to process fresh carrot juice providing a safe product without affecting

physicochemical characteristics.

1. Introduction

Thermal processing is the most common method of juice pasteurization. This process is effective

on the inactivation of pathogens, including E. coli present in the juices, but the high temperature

may cause losses of bioactive compounds and sensory attributes (Alighourchi and others 2014).

Novel food processing technologies have been focusing on offering microbiologically safe

products while retaining bioactive compounds and sensory attributes (Lee and Feng 2011). For

this purpose, emerging nonthermal technologies are under exploration, including ultrasound, high

pressure processing, pulsed electric fields, irradiation, and cold plasma for the preservation of fresh

fruits and vegetable products (Knorr and others 2011). Fresh fruit and vegetable products have

been associated with various foodborne outbreaks over the past several decades. E. coli serotypes

O157:H7 is one of the major causes of the epidemic in fruits and vegetable products, including the

juices (Besser and others 1993; CDC 1996; Cody and others 1999; Gillespie 2016). Although there

is no recent outbreak associated with E. coli in carrot juice, there is a chance for growth of this

pathogen in carrot juice and other low-acid juices. The HACCP regulation requires that the

processing conditions for juice pasteurization should reduce the most resistant microorganisms of

concerns by at least 5 log (US FDA, 2001).

Ultrasound processing has shown promising results in microbial inactivation in fruit and

vegetable juices (Knorr and others 2004) where the cavitation phenomenon generated by

ultrasound is responsible for microbial inactivation. During cavitation, there is a generation of

bubbles and micro-currents which can catalyze chemical reactions and disrupt microbial cells, lead

103

to the formation of wrinkles, ruptures, and perforation, which ultimately cause the death of the

microbial cells (Barbosa-Cánovas and Rodríguez 2002; Ugarte‐Romero and others 2006).

The effects of ultrasound on the inactivation of microorganisms and physicochemical

properties have been studied on many acidic fruit juices such as apple, orange, and strawberry

(Tiwari and others 2009; Patil and others 2009). Previous studies suggest that ultrasound is

successful in pasteurizing high acid juices. Processing of low-acid juices is challenging as a

number of pathogenic and spoilage microorganisms are resistant at high pH. Ultrasound processing

alone (sonication) is not very efficient on the inactivation of microorganisms in low-acid products;

therefore, it is generally combined with temperature (thermosonication) (Piyasena and others

2003). Ultrasound processing at sub-lethal temperatures (<50°C) requires either high acoustic

energy density (AED) or very long treatment times to get 5 log reduction of vegetative

microorganisms (Lee and others 2009). Many studies show that the combination of ultrasound and

temperature below 50°C is not very effective on micorbial inactivation (Raso and others 1998;

López-Malo and others 1999; Villamiel and de Jong, 2000) whereas the combination of ultrasound

and lethal temperatures has shown to have a strong synergistic effect in microbial load reduction

(Piyasena and others 2003; Feng and others 2008). Wordon and others (2012) found a significant

decrease in D-value of S. cerevisiae by ultrasound processing at 60°C (D-value 0.73 min) as

compared to temperature alone (D-value 3.53 min) and ultrasound alone (D-value 3.1 min). Lee

and others (2009) studied the inactivation of E. coli K12 using ultrasound combined with thermal

energy. They observed that the combination of lethal parameters (temperature and/or pressure)

significantly reduced the time required to obtain 5 log reduction. There is a temperature upper limit

that can be used in combination with ultrasound; when ultrasound is carried out at high

temperatures there is a cushioning effect, and the vapor filled bubbles break less violently, and the

104

effect of ultrasound in the combined process will be less significant (Ugarte-Romero and others

2007). Raso and others (1998) found that in the combination of ultrasound and high temperature

(>58°C), the level of reduction in microorganisms by temperature alone is similar to the one

achieved by the combined effect of ultrasound and temperature.

The objective of this work was to investigate the synergistic effect of ultrasound when

combined with mild temperatures on the inactivation of E. coli in fresh carrot juice and to

investigate possible changes in some of its physicochemical characteristics. In this study, carrot

juice was selected as a model food based on its pH (low-acidic; pH: 6.82) which could act as an

excellent medium for the growth of foodborne pathogens. Since the present study is focused on

the mild-pasteurization of fresh carrot juice; a surrogate for pathogenic E. coli was selected to

study the inactivation kinetics, unlike severe-pasteurization where the process is focused on the

inactivation of non-proteolytic Clostridium botulinum type E spores (Vervoort and others 2012).

This study identifies processing conditions to effectively pasteurize low-acid juices in short

periods of time making the process energy efficient and cost effective by combining ultrasound

and mild temperatures.

2. Materials and methods

2.1 Preparation of carrot juice

Fresh carrots (Dacus carota) were purchased in bulk from a local market in Pullman, Washington,

USA. Carrots were screened, washed, cut into pieces, and extracted by using a domestic juice

extractor (Hamilton Beach JE2200B, Black & Decker, MD, USA). The juice was then filtered

105

using cheesecloth to remove coarse particles and to make the juice similar to the one available in

the market. Prepared juice has the pH of 6.82 and total soluble solids of 8.5°Bx.

2.2 Culture preparation

The freeze-dried culture of E. coli ATCC 11775 strain was procured from the American Type

Culture Collection (Manassas, VA). The selection of this strain is based on the higher heat

resistance as compared to pathogenic strain E. coli O157:H7 (Gurtler and others 2010). Inoculation

study was carried out according to the method described by Moody and others (2014). The culture

was rehydrated with sterile Nutrient Broth (Difco, Becton, Dickinson & Company, Sparks, MD),

and inoculated into 100 mL of nutrient broth under continuous agitation (218 rpm, 37°C) until the

early stationary phase was reached. The absorbance of inoculated broth was read every hour at

562 nm using a spectrophotometer (Hewlett–Packard, Palo Alto, CA) and pour-plating was done

using Eosin Methylene Blue (EMB) Agar (Neogen©, Lansing, MI). A stock culture was prepared

using a sterile 20% glycerol solution and stored at −21°C until used.

2.3 Ultrasound treatment

A 400 W Hielscher® ultrasonic device (Hielscher USA Inc., Ringwood, NJ) with a probe of 22

mm, frequency of 24 kHz and amplitude of 120 μm was used in this study. Carrot juice sample

(500 mL) was placed into the 500 mL glass vessel (double jacket) connected to ultrasound

equipment and water bath (VWR Scientific, Model 1166, Radnor, PA). The temperature was

monitored using a k-type thermocouple (Omega Engineering Inc., Stamford, CT). The probe was

immersed 3 cm in depth with respect to the juice surface. The juice was pre-treated with ultrasound

and heat for about two to three minutes to bring the temperature to the target one, and it was kept

106

constant. After reaching the target temperature, E. coli culture was inoculated to get the final

concentration of >5 log CFU/mL. Sample at time zero was taken out immediately after the

inoculation. Inoculated juice was treated by ultrasound in combination with three different

temperatures (50, 54, and 58°C) to study the combined effect of ultrasound and heat. The selection

of different temperatures was based on the effectiveness of the combined processes. Ultrasound

treatments at a temperature below 50°C had shown the lesser effect on microbial inactivation

whereas the effect of ultrasound in the combined process is less significant when the processing

temperature is more than 58°C (Raso and others 1998; Ugarte-Romero and others 2007; Lee and

others 2009). The ultrasonic intensity (UI) for each treatment was 37.87 W/cm2. A wide range of

processing times (0 to 10 min) was selected to study the kinetics of inactivation. Samples were

collected every minute and kept in sterile test tubes with 0.1% peptone water. The fresh, untreated

carrot juice was considered as a control sample, and the treatments were conducted in triplicates.

2.4 Microbiological analysis

Microbiological analysis was conducted as per the methods described by Moody and others

(2014). Serial dilutions of the samples were made with 0.1% (w/v) peptone water (Becton,

Dickinson, and Company, Sparks, MD). One milliliter of decimal dilutions of processed juice was

pipetted into Petri dishes followed by addition of about 20 mL of molten eosin methylene blue

(EMB) agar (45°C) to each plate and was allowed to solidify for 30 min at room temperature. The

plates were turned upside down and incubated at 37ºC for 48 hours. Survivor counts were carried

out manually and expressed as CFU/mL.

107

2.5 Microbial inactivation kinetics

Three different general equations: first-order, Weibull distribution, and biphasic were selected to

model the observed inactivation data of E. coli. The models performance were evaluated based on

the statistical indices: correlation coefficient (R2 and pseudo-R2), root mean square error (RMSE),

F-value (compared to F critical), Bias factor (Bf), and Accuracy factor (Af) (Baranyi and other

1999). The value of R2 close to 1 and a low value of RMSE indicates the goodness of fit of the

model. The predicted values of the mathematical models and statistical indices were calculated

using Microsoft Excel 2016 and GInaFiT (Geeraerd and others 2005).

2.5.1 First-order

A first-order decay reaction usually describes the microbial inactivation kinetics in conventional

thermal pasteurization. This model is based on the assumption that all cells have equal resistance

to the treatment. Bigelow (1921) represents this model in the form of thermal death time and

decimal reduction time:

Log (Nt

N0) = −

t

D , (1)

where Nt is the survivor at time t (minutes), N0 is the initial microbial load, and D is the decimal

reduction time in minutes.

2.5.2 Weibull model

The Weibull model has two parameters with the assumption that microbial cells have different

resistance to the applied treatment. This model was initially proposed by Peleg and Cole (1998) in

108

the form a Weibull distribution function which was later modified by Mafart and others (2002)

and again reparametrized by van Boekel (2002) (Equation 2).

Log (Nt

N0) = − (

t

δ)

p

, (2)

where δ is the time for 1 log reduction and p describes the shape of survival curve (linear: p=1;

nonlinear: p≠1; upward concavity: p<1; downward concavity: p>1 (van Boekel 2002).

2.5.3 Biphasic model

A biphasic model that was proposed by Cerf (1977) can describe nonlinear microbial death curves.

According to this model, the microbial population is categorized into two different populations:

treatment-sensitive and treatment-resistant. Geeraerd and others (2005) expressed this model in

the following form:

𝑁(𝑡) = 𝑁0(𝑓𝑒−𝑘1 𝑡 + (1 − 𝑓)𝑒−𝑘2 𝑡), (3)

where f and (1-f) are the fractions of treatment-resistant and treatment-sensitive cells, respectively.

The parameters k1 and k2 are death rate constant for the two phases.

2.6 Determination of color parameters

Color was measured using the CIE (Commission Internationale d'Eclairage) color scale to

measure the parameters L* (lightness to darkness), a* (redness to greenness) and b* (yellowness

to blueness). Juice sample (40 mL) was placed into high-density polyethylene bags, and the color

parameters were determined using a Minolta CM-2002 colorimeter (Minolta Camera Co., Osaka,

Japan).

109

2.7 Determination of pH, soluble solids content, and acidity

The pH of fresh and processed samples immediately after treatment were measured by using a pH

meter (Thermo Electron Corporation, Orion 4 Star). Five milliliters of the sample were poured into

a beaker, stirred, and the electrode was immersed directly into the sample at 20ºC. The soluble

solids content was assessed in the fresh and processed juice using a refractometer (Atago No. 3840,

Atago Co., Ltd, Japan). A sample of the juice was placed on the prism of the refractometer and

read directly. The measurements were done in triplicate, and the obtained values were reported as

ºBrix.

For the determination of acidity, juice sample (5 mL) was titrated against a standardized

NaOH solution. Titratable acidity (TA) was expressed in terms of citric acid.

2.8 Determination of phenolic compounds, ascorbic acid, and total carotenoid

Total phenolic compounds were determined using a colorimetric method described by Singleton

and others (1999) with some modification. This technique is based on the reduction of the Folin-

Ciocalteau reagent by phenolic compounds with a formation of a blue complex. The phenolic

compounds were extracted from 1 mL of carrot juice with 10 mL of methanol for 24 h in darkness

under agitation and separated by centrifugation at 4500 rpm for 10 min using an MSE Centaur 2

centrifuge (MSE (UK) Ltd. London, UK). A 40 μL aliqot of extracted solution, 130 μL of Folin-

Ciocaltaeau reagent, and 625 μL of deionized water were mixed using a vortex (Daigger Vortex

Genie 2 Model G-560, Daigger Scientific, Inc, Vermon Hills, IL) and left at room temperature

(22°C ± 1°C) for 5 min. After that, 1.25 μL of saturated sodium carbonate (Na2CO3) solution

(20%) was added, and the mixture was left for 90 min in darkness. The absorbance was measured

110

at 760 nm using a spectrophotometer (Spectronic 20 Genesys, Spectronic Instruments Inc.,

Rochester, NY). The total phenolic compounds were measured from the calibration curve obtained

from standard gallic acid solution. Total phenolic compounds were expressed as μg of gallic acid

equivalents (GAE) per mL of carrot juice.

The ascorbic acid content of the juice before and after treatment was determined using the

method described by Helrich (1990), which is based on the 2, 6-dichlorophenol-indophenol

titration. Carrot juice sample (5 mL) was placed in a centrifuge tube and 10 mL of metaphosphoric

acid-acetic acid solution was added. The mixture was centrifuged at 4000 rpm for 5 min and 10

mL of supernatant was titrated with indophenol solution to get faint pink color end point.

The total carotenoid content was measured in fresh and processed carrot juice with the

methodology described by Zhou and others (2009) with some modifications. Carrot juice samples

(2 mL) was mixed with 10 mL chloroform/methanol (2:1). After mixing, the organic phase was

separated from the aqueous phase. Repeated extraction of the aqueous phase was done until it was

colorless. The separated organic phase was collected and diluted to 50 mL using

chloroform/methanol. Absorbance was measured at 450 nm, and results were expressed as μg β-

carotene equivalent/100 mL of carrot juice.

2.9 Statistical analysis

Data obtained was represented as mean value ± standard deviation (SD). One-way ANOVA was

conducted at a significance level of 0.05. Significant differences between mean values were

determined by LSD (least significant differences) pairwise comparison test. Statistical analysis

was performed using SAS 9.2 (SAS Institute Inc. NC, USA).

111

3. Results and discussion

3.1 E. coli Inactivation

Applied processing temperatures (50, 54, and 58°C) and treatment times (0 to 10 min) both had a

significant effect (p<0.05) on E. coli inactivation (Figure 18). More than 5 log reduction of E. coli

was achieved in 2 min by the application of ultrasound (24 kHz, 120 µm, 400 W) at 58°C. In the

treatment at 54°C, 10 min was required to achieve the same level of reduction. However, at 50°C,

only 3.5 log reduction was achieved after 10 min of treatment.

The cavitation effect due to application ultrasound might have caused the dispersion of the

microbial clumps and disruption of cell membranes, which is responsible for lysis of

microorganisms (Hayer 2010; Cruz-Cansino and others 2016). The application of mild heat (50,

54, and 58°C) helped to weaken the cell membrane which further supported the cavitation

phenomenon for the disruption of the cell membrane causing the death of microbial cells more

efficiently (Villamiel and de Jong 2000). In addition to applied heat, during ultrasound processing,

there is also the formation of hydrogen peroxide and free radicals which also enhances the

inactivation of microorganisms (Ciccolini and others 1997; Cruz-Cansino and others 2016).

At the beginning of the treatment, there was a shoulder in the inactivation curve for the

treatments at 50 and 54°C. The shoulder might be due to the formation of clumps by E. coli, where

the treatment conditions were too weak to break them (Hayer 2010; Cruz-Cansino and others

2016). For the ultrasound treatment at 58°C, there was no shoulder, as at this temperature level

treatment dose might be strong enough to break the microbial clumps and in this case, there was

fast inactivation of E. coli, which was followed by tailing which it was also observed in the

112

treatments at 50 and 54 °C. The presence of treatment-resistant subpopulations as well as the

presence of any ‘artifact’ that interfere the inactivation of microorganisms can cause tailing of the

survivor curve (Bermúdez-Aguirre and others 2009).

In a recent study, Lee and others (2013) found 5 log reduction of E. coli K12 in apple cider

using an ultrasound treatment (3 W/mL, 59°C) for 3.8 min; the time required for 5 log reduction

in their studies was slightly higher than the present work. Cruz-Cansino and others (2016) studied

the effect of amplitude (60-90%) and treatment time during ultrasound treatments (1500 W,

20 kHz) on the inactivation of E. coli in cactus pear juice. They observed that the treatment time

of 5 min at 90% level of amplitude resulted in inactivation below the detectable limit (7 log

reduction). In another study, 5.07 log reduction of E. coli O157:H7 was obtained in apple cider by

combined application of ultrasound (20 kHz, 118 W/cm2) and temperature (57±1°C) for 18 min,

whereas temperature alone rendered only 3.79 log reduction (D'amico and others 2006). These

results show a strong synergistic effect between ultrasound and temperature on the inactivation of

microorganisms. Jabbar and others (2014) found only 1.5 and 1.8 log reduction of the total plate

count, and yeast and molds respectively in carrot juice by sonication (20 kHz, 70% amplitude,

15°C) in 5 minutes. The lower level of inactivation in their work might be due to the application

of ultrasound at low temperature and low amplitude as compared to the present one. Other

researchers (Ugarte-Romeo and others 2006; Lee and others 2009) have used a different strain of

E. coli; this might be one of the reasons for different results. Including the strain of

microorganisms, there are also other factors that influence the inactivation such as the size of the

treatment vessels, the position of the sonotrode, acoustic energy density, and the amplitude of the

applied frequency.

113

Hence, the results show increasing the treatment temperature enhances the rate of

inactivation proving synergistic effect between ultrasound and temperature. However, increasing

the processing temperature beyond certain limits, the role of cavitation becomes less relevant. The

maximum temperature that can be used in ultrasound processing depends on the media as it is

known that increasing the temperature will increase the vapor pressure and decrease surface

tension. This phenomenon will lead to the collapse of the cavitation bubbles less violently which

will have less impact on microbial inactivation (Ugarte-Romero and others 2007).

Figure 18. Inactivation kinetics of E. coli: (a) inactivation curve at different temperatures (50, 54, and

58°C); Modeling the inactivation kinetics of E. coli by ultrasound at 50 (b), 54 (c), and 58°C (d) using first-

order, Weibull, and Biphasic model.

-6

-5

-4

-3

-2

-1

0

0 2 4 6 8 1 0

Log

(Nt/

N0)

Time (min)a

US+50C US+54C US+58C

-4

-3

-2

-1

0

0 2 4 6 8 10

Log

(Nt/

N0)

Time (min)b

Observed First-Order

Weibull Biphasic

-5

-4

-3

-2

-1

0

0 2 4 6 8 10

Log

(Nt/

N0)

Time (min)c


Weibull Biphasic

-6

-4

-2

00 0.4 0.8 1.2 1.6 2

Log

(Nt/

N0)

Time (min)d


Weibull Biphasic

114

3.2 Mathematical modeling

Survival curves of E. coli obtained by plotting log (Nt/N0) vs. treatment time (min) at different

temperature were fitted to first-order, Weibull, and biphasic models. The first-order model has

been widely used to study the inactivation curves in many thermal and nonthermal processes. In

the present study, the observed data on E. coli inactivation did not fit well with the first-order

model as it can be observed in Figure 18, as well as from statistical indices (Table 1) at various

temperature levels as compared to the other models. Regarding statistical indices, for the first-

order model, the value of R2 was low as compared to the other two models. The lower value of R2

indicates comparatively lower goodness of fit. Furthermore, the value of RMSE was high as

compared to the other two models; this also supports that first-order model did not have good

correlation with the inactivation kinetics data. F-test shows the calculated value of F was lower

than the critical value in all the models and at all processing conditions; but when three models

were compared the value of F was higher in the first-order model. This kind of deviation of the

inactivation curve from the conventional first-order model was well described in ultrasound as

well as in other nonthermal processing of fruits and vegetable juices as shown by Peleg and Cole

1998; Peleg and Cole 2000; Lee and others 2009; Adekunte and others 2010b. The results of the

present study are in line with the finding of Lee and others (2013) who observed similar deviation

of the inactivation curve. This kind of deviation from the linear first-order model in ultrasound can

be explained by the vitalistic approach (Cerf 1977; Cole and others 1993). According to the

vitalistic concept, in a microbial population, individual cells have different resistance to the lethal

treatment, which results into nonlinear inactivation curves with shoulders and tails (Cole and

others 1993). In the present study, treatment-sensitive cells of E. coli most likely inactivated first,

and the remaining cells with higher resistant to combined treatment were slowly inactivated

115

resulting in a tailing effect. The D-value (time required for one log reduction of the microbial

population) for the ultrasound treatment at 50, 54, and 58°C was found to be 2.95, 2.42, and 0.44

min respectively. In a study conducted by Lee and others 2009, there was a negligible reduction

of E. coli K12 cell at 54°C, 4 min; but when ultrasound was introduced at same temperature and

time, there was about 4 log reduction. This shows that application of ultrasound significantly

reduces the time required for 5 log reduction. Regarding the treatment at 58°C in the present study,

2.2 min was required to achieve 5 log reduction of E. coli in carrot juice. It can be considered as a

considerable reduction in the processing time as compared to a conventional thermal treatment,

especially for the low-acid juices, which require a higher temperature and a longer treatment time

if heat is used alone.

The Weibull model was successful in fitting E. coli inactivation data obtained after

combined treatments, as can be seen from the kinetic curves (Figure 18) and statistical indices

values (Table 9). The value of shape factor (p = 0.33 to 0.58) shows that the inactivation curves

have upward concavity or tailing. The formation of this concavity is mainly due to fast inactivation

of sensitive cells of the microbial population and slow inactivation of resistant cells. (Peleg 2006,

Feng and others 2008). Ugarte-Romero and others (2006) observed tailing (upward concavity)

with p-values from 0.547 to 0.720 during E. coli K12 inactivation by the combined application of

ultrasound (20 kHz) and heat (40-60°C) in apple cider, which is very similar to the current results.

On the other hand, Adekunte and others 2010b observed downward concavity during yeast

inactivation in tomato juice by ultrasound (1500 W, 61 µm amplitude, 20 Hz and 25-39.9°C). The

time for one log reduction using Weibull model in the present study was found to be 2.79, 2.10,

0.27 min for the ultrasound treatment at 50, 54, and 58°C respectively. Considering the treatment

116

at 58°C, according to the Weibull model, the time required for 5 log reduction of E. coli in carrot

juice is 1.35 min at the selected processing conditions.

For some treatments, there was fast inactivation at the beginning followed by slow

inactivation, forming two phases in the curve. To fit the inactivation curve with two phases, the

biphasic model was further taken into consideration. Nonlinear inactivation curve with two phases

were fitted well with the biphasic model as demonstrated by slightly high values of pseudo-R2 and

relatively low RMSE value (Table 9) as compared to Weibull. Regarding the F-test, there was no

significant difference between the Weibull and biphasic models. Results show that with the

increase in ultrasound processing temperature, faster inactivation of E. coli was observed at the

beginning of the treatment. For example, in an ultrasound treatment at 58°C, there was no shoulder

(Figure 18d). The biphasic model fitted slightly better the processing condition at 58°C than the

ones at 50°C and 54°C as it is observed by pseudo-R2 and RMSE-values. The time for 5 log

reduction using the biphasic model in the present study was found to be 14.15, 10.6, 1.35 min for

the ultrasound treatment at 50, 54, and 58°C respectively. Some other studies also show similar

trends in inactivation by ultrasound (Ugarte-Romero and others 2006; D’Amico and others 2006).

The bias factor values for all the treatment were close to one (0.998-1.094) except for the

first-order model (treatment at 58°C, Bf 0.745, see Table 9). Bf values close to one indicates good

agreement between observed and predicted values, whereas the value of Bf smaller than one

indicates under prediction and the value greater than one indicates over prediction. At all treatment

conditions, the biphasic model and the Weibull model had lower accuracy factors (Af 1.028-1.869)

as compared to first-order model (Af 1.789-1.979). The lower the value of accuracy factor the

better is the model. Hence, biphasic and Weibull model could accurately predict the survivors

117

during ultrasound treatment at mild temperatures. Ultrasound treatment at a temperature below

54°C has shown to have a shoulder in the inactivation curve; in this case, Weibull model would be

an excellent tool to predict the survivors. For ultrasound treatments at 58°C, there was no shoulder

formation. Instead, there are two stages in the inactivation curve, therefore the biphasic model

would adequately predict the survivors.

Table 9. Statistical comparison of three different mathematical models: First-order, Weibull, and Biphasic

based on R-square, Root mean square error (RMSE), Bias factor (Bf), Accuracy factor (Af), and F-test.

Processing

Conditions Statistical Index

Models

First-order Weibull Biphasic

US+50°C

R-Square 0.915 0.973 0.989

RMSE 0.415 0.255 0.179

Bias factor (Bf) 1.014 1.094 1.046

Accuracy factor (Af) 1.832 1.572 1.272

F 1.092 1.027 1.006

F table 3.787 3.787 3.787

US+54°C

R-Square 0.900 0.943 0.964

RMSE 0.554 0.459 0.409

Bias factor (Bf) 1.019 1.093 1.047


F 1.110 1.06 1.032

F table 3.787 3.787 3.787

US+58°C

R-Square 0.837 0.995 0.999

RMSE 1.191 0.259 0.095

Bias factor (Bf) 0.745 0.998 0.999


F 1.194 1.003 1.000

F table 6.388 6.388 6.388

US: Ultrasound

3.3 Effects of treatment on pH, soluble solids content, and acidity

The pH, soluble solids, and acidity of the juice are associated with the sensory quality, therefore

changes in these three attributes might negatively affect sensory. In this study, there were no

118

significant changes (p>0.05) in pH, soluble solids content or acidity of carrot juice before and after

ultrasound processing (Table 10). In some studies, ultrasound treatments had caused significant

changes in pH of fruit juices (Bermúdez-Aguirre and Barbosa-Cánovas 2012). The change in pH

during ultrasound treatments could be due to the generation of hydrogen peroxide or nitrate and

nitrite in the aqueous medium as observed by Supeno (2000). Another study on grape juice shows

no significant changes in pH, soluble solids and acidity after ultrasound treatments (20 kHz, 1500

W) at 32 to 45°C (Tiwari and others 2010). The results for soluble solids from the present studies

are in line with the observation made by various researchers in different products: grapefruit juice

(Aadil and others 2013), orange juice (Tiwari and others 2008), and Kasturi lime juice (Bhat and

others 2011).

Table 10. Physicochemical parameters of carrot juice before and after processing

Sample pH °Brix

Titratable

Acidity (%)

Phenolic

Compounds (µg/ml)

Ascorbic Acid

(mg/100ml)

US+50°C 6.80±0.01 a 8.0±0.1 a 0.29±0.01 a 20.25±0.91 a 4.8±0.25 a

US+54°C 6.82±0.01 a 8.3±0.1 a 0.30±0.01 a 20.38±0.61 a 4.8±0.25 a

US+58°C 6.82±0.01 a 8.4±0.1 a 0.30±0.01 a 20.19±0.61 a 4.8±0.25 a

Control 6.82±0.01 a 8.5±0.2 a 0.30±0.01 a 20.63±0.60 a 4.8±0.00 a

Values with different letters in the same column are significantly different (p<0.05)

US: Ultrasound

119

3.4 Effects of treatment on color parameters

There was no significant difference (p>0.05) in color parameters (L*, a* and b*) between fresh

and ultrasound processed carrot juice (Table 11). Carotenoids are responsible for the characteristic

color of carrot juice. It clearly indicates that there was no degradation of carotenoids, and therefore,

there were no changes in a* (redness) and b* (yellowness) parameters. Maillard reaction can occur

in the juice resulting in darker color thereby reducing the lightness (L* value), but in the present

study, it was not observed. Valero and others (2007) found similar results in orange juice, where

there were no changes in color after sonication at 23-500 kHz and 120-600 W for 0 to 180 minutes.

However, Tiwari and others (2008) found a significant (p<0.05) decrease in a* value and increase

in L* and b* value when orange juice was treated by ultrasound (22.79 W/cm2) for 10 min. Cheng

and others (2007) observed the color change in sonicated and carbonated guava juice, i.e., the

authors found significant differences in all the color properties, although the naked eye did not

distinguish those differences. Fonteles and others (2012) concluded that during sonication,

disruption of cell membranes causes the formation of carotenoid-protein complexes which results

in color change. In the present study, the heat treatment (≤58°C) was moderate, so there were no

significant (p>0.05) effects on the carrot juice color.

Table 11. Color parameters of carrot juice before and after processing

Sample L* a* b*

US+50°C 50.94±1.73 a 18.83±1.10 a 33.10±0.81 a

US+54°C 50.10±0.68 a 19.51±0.07 a 32.96±0.20 a

US+58°C 50.81±1.73 a 20.66±2.59 a 31.75±1.76 a

Control 50.05±0.68 a 19.33±1.12 a 32.71±0.90 a

120

Values with different letters in the same column are significantly different (p<0.05)

US: Ultrasound

3.5 Effects of treatment on total carotenoid

There was non-significant (p>0.05) increase in total carotenoid content in carrot juice after

processing (Figure 19). Ultrasound processing at 50, 54, and 58ºC resulted in an increase in total

carotenoid content, about 2.71%, 3.27%, and 3.44%, respectively as compared to fresh juice. The

mechanical disruption of cell walls during cavitation might have released free carotenoid in juice,

resulting into its slight increment. Within the applied three different temperatures, the total

carotenoid contents increased with the increase in processing temperature (Figure 2). This increase

might be attributed to the release of bound form of carotenoid due to the combined effect of

ultrasound and heat. The incorporation of heat during the process might cause the softening of the

vegetable tissues thereby supporting the release of carotenoid during cavitation. Abid and others

(2014) observed a significant increase in total carotenoid content in apple juice treated with

ultrasound for 30 and 60 min as compared to the untreated sample. In the present study, the

treatment times were much shorter than in other studies which resulted in a minor increase in total

carotenoid content (1777.5 µg/100 ml in fresh juice to 1841.5 µg/ 100 ml in the juice sonicated at

58°C).

121

Figure 19. Carotenoid content of juice before and after processing, control indicates the fresh juice

without any treatment, US+50°C, US+54°C, US+58°C are three different ultrasound treatment at 50, 54

and 58°C respectively. There was non-significant (p>0.05) increment in total carotenoid content after

each treatment.

3.6 Effects of treatment on phenolic compounds

Total phenolic compounds did not change significantly (p>0.05) when ultrasound was applied at

50, 54 and 58°C for 10 min. The fresh juice had 20.63 g/mL of phenolic compounds and juice

processed at 50, 54, and 58ºC presented values of 20.25, 20.38 and 20.19 g/mL, respectively.

Abid and others (2014) observed a significant increase in phenolic compounds in apple juice after

being treated by ultrasound for 30 and 60 minutes.

The increase in phenolic compounds during ultrasound might be due to disruption of cell

walls, promoting the release of intracellular components. Furthermore, this increase in phenolic

compounds could also be justified by the reaction between hydroxyl radicals generated during

sonication, and the aromatic ring of polyphenols (Ashokkumar and others 2008; Zafra-Rojas and

others 2013). It seems that processing for a longer time (20 and 30 minutes) causes these changes,

1710

1760

1810

1860

Control US+50°C US+54°C US+58°C

Car

ote

no

ids

Co

nte

nt

(µg/

10

0 m

l)

a

a a a

122

and therefore the phenolic compounds are released. In this study, the processing time was only 10

minutes, so there were no structural modifications that would quantify a higher content of phenolic

compounds.

3.7 Effects of treatment on ascorbic acid

There was no significant change (p<0.05) in ascorbic acid content in carrot juice before and after

ultrasound processing at the selected conditions (Table 10). All samples had a content of ascorbic

acid of 4.80 mg/100 mL of carrot juice. Several reports show an increase in the content of ascorbic

acid after ultrasound processing. However, in those studies, ultrasound was applied at room

temperature with longer processing time as compared to the present study. For example, Zou and

Jiang (2016) processed carrot juice by ultrasound (40 kHz, 0.5 W/cm2) for 20 or 40 min. These

authors found 3.61% and 8.17% increase in ascorbic acid content as compared to fresh juice when

processed for 20 minutes and 40 minutes, respectively. Another study conducted by Aadil and

others (2013) reported significant (p<0.05) increase in ascorbic acid content for sonicated apple

juice treated at 60 and 90 min, while there was no change in its content for the same juice in 30

min of treatment. Abid and others (2014) showed a significant increase (p<0.05) in ascorbic acid

content in the samples processed for 60 and 90 min in comparison to samples treated for 30 min.

However, there are studies in which the authors found a decrease in the content of ascorbic acid

after ultrasound treatment. Khandpur and Gogate (2015) treated carrot juice with ultrasound (20

kHz, 100 W) for 15 min; the authors found a reduction of 39.5% in its ascorbic acid content as

compared to the fresh juice. Saeeduddin and others (2015) found 100% retention in ascorbic acid

content of pear juice after ultrasound processing (750 W, 20 kHz, 70% amplitude) at 25°C, 10

min. However, with increasing processing temperature to 45 and 65°C at the same treatment time,

123

the ascorbic acid content decreased by 6.25 and 8.75%, respectively. This indicates that ascorbic

acid can be retained more when the ultrasound is performed at low temperatures. Ascorbic acid is

very sensitive to heat, and it degrades as the processing temperature and time is increased.

Santhirasegaram and others (2013) found a significant (p<0.05) decrease in the ascorbic acid

content of mango juice treated by either ultrasound or thermal treatment as compared to fresh juice.

Adekunte and others (2010a) suggest that ascorbic acid degrades in fruits and vegetable juice after

ultrasound treatments. However, the changes in ascorbic acid depend on the intensity of the

ultrasound and the processing time. Thus, it is important to take into account the temperature and

time to preserve ascorbic acid during ultrasound processing.

4. Conclusions

This study shows the positive effects of using ultrasound in combination with mild temperatures

to process carrot juice fulfilling the FDA standards for juice pasteurization. The best processing

condition for carrot juice is 58°C for 2 min (24 KHz/120 µm, 37.87 W/cm2), which renders 5-log

reduction of E. coli at the shortest time without affecting the physicochemical quality and bioactive

compounds. Mathematical modeling of the survivor’s curve shows both Weibull and biphasic

model to be a good fit to predict the inactivation kinetics. All selected processing conditions had

no significant effect on the quality attributes of the juice. This study suggests that the effective

combination of ultrasound and mild temperature could be used to pasteurize low-acid juices at a

shorter time as compared to thermal processing and without significantly affecting the


124

References

Aadil RM, Zeng X-A, Han Z, Sun D-W. 2013. Effects of ultrasound treatments on quality of

grapefruit juice. Food Chem 141(3):3201-6.

Abid M, Jabbar S, Hu B, Hashim MM, Wu T, Lei S, Khan MA, Zeng X. 2014. Thermosonication

as a potential quality enhancement technique of apple juice. Ultrason Sonochem 21(3):984-

90.

Adekunte A, Tiwari B, Cullen P, Scannell A, O’Donnell C. 2010a. Effect of sonication on colour,

ascorbic acid and yeast inactivation in tomato juice. Food Chem 122(3):500-7.

Adekunte A, Tiwari B, Scannell A, Cullen P, O'Donnell C. 2010b. Modelling of yeast inactivation

in sonicated tomato juice. Int J Food Microbiol 137(1):116-20.

Alighourchi H, Barzegar M, Sahari MA, Abbasi S. 2014. The effects of sonication and gamma

irradiation on the inactivation of Escherichia coli and Saccharomyces cerevisiae in

pomegranate juice. Iran J Microbiol 6(1):51.

Ashokkumar M, Sunartio D, Kentish S, Mawson R, Simons L, Vilkhu K, Versteeg CK. 2008.

Modification of food ingredients by ultrasound to improve functionality: a preliminary

study on a model system. Innovative Food Sci Emerging Technol 9(2):155-60.

Baranyi J, Pin C, Ross T. 1999. Validating and comparing predictive models. Int J Food

Microbiol 48(3):159-66.

Barbosa-Cánovas G, Rodríguez J. 2002. Update on nonthermal food processing technologies:

Pulsed electric field, high hydrostatic pressure, irradiation and ultrasound. Food Aust

54(11):513-20.

125

Bermúdez-Aguirre, D, Corradini, M. G, Mawson, R, & Barbosa-Cánovas, GV. (2009). Modeling

the inactivation of Listeria innocua in raw whole milk treated under thermo-

sonication. Innovative Food Sci Emerging Technol 10(2):172-78.

Bermúdez-Aguirre, D., & Barbosa-Cánovas, GV. (2012). Inactivation of Saccharomyces

cerevisiae in pineapple, grape and cranberry juices under pulsed and continuous thermo-

sonication treatments. J Food Eng, 108(3): 383-392.

Besser RE, Lett SM, Weber JT, Doyle MP, Barrett TJ, Wells JG, Griffin PM. 1993. An outbreak

of diarrhea and hemolytic uremic syndrome from Escherichia coli O157:H7 in fresh-

pressed apple cider. Jama-J Am Med Assoc 269 (17):2217-20.

Bhat R, Kamaruddin NSBC, Min-Tze L, Karim A. 2011. Sonication improves kasturi lime (Citrus

microcarpa) juice quality. Ultrason Sonochem 18(6):1295-300.

Bigelow, WD. 1921. The logarithmic nature of thermal death time curves. J Infect Dis 528-36.

CDC 1996. Outbreak of Escherichia coli O157:H7 infections associated with drinking

unpasteurized commercial apple juice. British Columbia, California, Colorado, and

Washington Morbid Mortal Wkly Rep 45 (44):975.

Cerf O. 1977. A review. Tailing of survival curves of bacterial spores. J Appl Bacteriol 42(1):1-

19.

Cheng L, Soh C, Liew S, Teh F. 2007. Effects of sonication and carbonation on guava juice quality.

Food Chem 104(4):1396-401.

126

Ciccolini L, Taillandier P, Wilhem A, Delmas H, Strehaiano P. 1997. Low frequency thermo-

ultrasonication of Saccharomyces cerevisiae suspensions: effect of temperature and of

ultrasonic power. Chem Eng J 65(2):145-9.

Cody SH, Glynn MK, Farrar JA, Cairns KL, Griffin PM, Kobayashi J, Fyfe M, Hoffman R, King

AS, Lewis JH. 1999. An outbreak of Escherichia coli O157:H7 infection from

unpasteurized commercial apple juice. Ann Intern Med 130(3):202-9.

Cole MB, Davies KW, Munro G, Holyoak CD, & Kilsby, DC. 1993. A vitalistic model to describe

the thermal inactivation of Listeria monocytogenes. J Ind Microbiol, 12(3-5):232-39.

Cruz-Cansino NDS, Reyes-Hernández I, Delgado-Olivares L, Jaramillo-Bustos DP, Ariza-Ortega,

JA, Ramírez-Moreno E. 2016. Effect of ultrasound on survival and growth of Escherichia

coli in cactus pear juice during storage. Braz J Microbiol, 47(2):431-437.

D'amico DJ, Silk TM, Wu J, Guo M. 2006. Inactivation of microorganisms in milk and apple cider

treated with ultrasound. J Food Prot 69(3):556-63.

Feng H, Yang W, Hielscher T. 2008. Power ultrasound. Food Sci Technol Int 14(5):433-6.

Fonteles TV, Costa MGM, de Jesus ALT, de Miranda MRA, Fernandes FAN, Rodrigues S. 2012.

Power ultrasound processing of cantaloupe melon juice: effects on quality parameters.

Food Res Int 48(1):41-8.

Geeraerd A, Valdramidis V, Van Impe J. 2005. GInaFiT, a freeware tool to assess non-log-linear

microbial survivor curves. Int J Food Microbiol 102(1):95-105.

Gillespie C. 2016. 15 E. coli Outbreaks of 2015. Food poisoning bulletin. Retrieved June 2, 2016,

from https://foodpoisoningbulletin.com/2016/15-e-coli-outbreaks-of-2015/

127

Gurtler JB, Rivera RB, Zhang HQ, Geveke DJ. 2010. Selection of surrogate bacteria in place of E.

coli O157:H7 and Salmonella Typhimurium for pulsed electric field treatment of orange

juice. Int J Food Microbiol 139(1):1-8.

Hayer K. 2010. The effect of ultrasound exposure on the transformation efficiency of Escherichia

coli HB101. Biosci Horiz 3(2):141-7.

Helrich, K. 1990. Official Methods of the Association of Official Analytical Chemists (AOAC).

Virginia USA: Association of Official Analytical Chemists.

Jabbar S, Abid M, Hu B, Muhammad Hashim M, Saeeduddin M, Lei S, Wu T, Zeng X. 2014.

Influence of sonication and high hydrostatic pressure on the quality of carrot juice. Int J

Food Sci Tech 49(11):2449-57.

Khandpur P, Gogate PR. 2015. Effect of novel ultrasound based processing on the nutrition quality

of different fruit and vegetable juices. Ultrason Sonochem 27:125-36.

Knorr D, Froehling A, Jaeger H, Reineke K, Schlüter O, Schoessler K. 2011. Emerging

technologies in food processing. Annual Rev Food Sci Technol 2:203-35.

Knorr D, Zenker M, Heinz V, Lee D-U. 2004. Applications and potential of ultrasonics in food

processing. Trends Food Sci Technol 15(5):261-6.

Lee H, Feng H. 2011. Effect of power ultrasound on food quality. In Feng H., Barbosa-Cánovas

GV., Weiss J. Editors. Ultrasound technologies for food and bioprocessing. New York:

Springer. p 559-82.

128

Lee H, Kim H, Cadwallader KR, Feng H, Martin SE. 2013. Sonication in combination with heat

and low pressure as an alternative pasteurization treatment-effect on Escherichia coli K12

inactivation and quality of apple cider. Ultrason Sonochem 20(4):1131-8.

Lee H, Zhou B, Liang W, Feng H, Martin SE. 2009. Inactivation of Escherichia coli cells with

sonication, manosonication, thermosonication, and manothermosonication: microbial

responses and kinetics modeling. J Food Eng 93(3):354-64.

López-Malo A, Guerrero S, Alzamora SM. 1999. Saccharomyces cerevisiae thermal inactivation

kinetics combined with ultrasound. J Food Prot 62(10): 1215-17.

Mafart P, Couvert O, Gaillard S, Leguérinel I. 2002. On calculating sterility in thermal

preservation methods: application of the Weibull frequency distribution model. Int J Food

Microbiol 72(1):107-13.

Moody A, Marx G, Swanson BG, Bermúdez-Aguirre D. 2014. A comprehensive study on the

inactivation of Escherichia coli under nonthermal technologies: High hydrostatic pressure,

pulsed electric fields and ultrasound. Food Control 37:305-14.

Patil S, Bourke P, Kelly B, Frías JM, Cullen PJ. 2009. The effects of acid adaptation on

Escherichia coli inactivation using power ultrasound. Innovative Food Sci Emerging

Technol 10(4):486-90.

Peleg M, Cole MB. 1998. Reinterpretation of microbial survival curves. Cr Rev Food Sci

38(5):353-80.

Peleg M, Cole MB. 2000. Estimating the survival of Clostridium botulinum spores during heat

treatments. J Food Prot 63(2):190-5.

129

Peleg M. 2006. Advanced quantitative microbiology for foods and biosystems: models for

predicting growth and inactivation. CRC Press.

Piyasena P, Mohareb E, McKellar R. 2003. Inactivation of microbes using ultrasound: a review.

Int J Food Microbiol 87(3):207-16.

Raso, J., Pagan, R., Condon, S., & Sala, F. J. (1998). Influence of temperature and pressure on the

lethality of ultrasound. Appl Environ Microbiol 64(2): 465-71.

Saeeduddin M, Abid M, Jabbar S, Wu T, Hashim MM, Awad FN, Hu B, Lei S, Zeng X. 2015.

Quality assessment of pear juice under ultrasound and commercial pasteurization

processing conditions. LWT-Food Sci Technol 64(1):452-8.

Santhirasegaram V, Razali Z, Somasundram C. 2013. Effects of thermal treatment and sonication

on quality attributes of Chokanan mango (Mangifera indica L.) juice. Ultrason Sonochem

20(5):1276-82.

Singleton VL, Orthofer R, Lamuela-Raventos RM. 1999. Analysis of total phenols and other

oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Method

Enzymol 299:152-78.

Supeno, PK. 2000. Sonochemical formation of nitrate and nitrite in water. Ultrason

Sonochem, 7(3):109-113.

Tiwari BK, Muthukumarappan K, O’Donnell C, Cullen P. 2008. Effects of sonication on the

kinetics of orange juice quality parameters. J Agr Food Chem 56(7):2423-8.

Tiwari BK, O’Donnell C, Cullen P. 2009. Effect of nonthermal processing technologies on the

anthocyanin content of fruit juices. Trends Food Sci Technol 20(3):137-45.

130

Tiwari, BK., Patras, A., Brunton, N., Cullen, PJ., & O’Donnell, CP. 2010. Effect of ultrasound

processing on anthocyanins and color of red grape juice. Ultrason Sonochem, 17(3):598-

604.

Ugarte‐Romero E, Feng H, Martin SE, Cadwallader KR, Robinson SJ. 2006. Inactivation of

Escherichia coli with power ultrasound in apple cider. J Food Sci 71(2): E102-E8.

Ugarte‐Romero E, Feng H, Martin SE. 2007. Inactivation of Shigella boydii 18 IDPH and Listeria

monocytogenes Scott A with power ultrasound at different acoustic energy densities and

temperatures. J Food Sci 72(4):M103-M107.

US FDA. 2001. United States Food and Drug Administration. Hazard analysis and critical control

point (HACCP); procedures for the safe and sanitary processing and importing of juices;

final rule, Fed Regist 66:6138-202.

Valero M, Recrosio N, Saura D, Muñoz N, Martí N, Lizama V. 2007. Effects of ultrasonic

treatments in orange juice processing. J Food Eng 80(2):509-16.

van Boekel MA. 2002. On the use of the Weibull model to describe thermal inactivation of

microbial vegetative cells. Int J Food Microbiol 74(1):139-59.

Vervoort L, Van der Plancken I, Grauwet T, Verlinde P, Matser A, Hendrickx, M, Van Loey A.

2012. Thermal versus high pressure processing of carrots: a comparative pilot-scale study

on equivalent basis. Innovative Food Sci Emerging Technol 15: 1-13.

Villamiel M, de Jong P. 2000. Inactivation of Pseudomonas fluorescens and Streptococcus

thermophilus in Trypticase® Soy Broth and total bacteria in milk by continuous-flow

ultrasonic treatment and conventional heating. J Food Eng 45(3): 171-79.

131

Wordon B, Mortimer B, McMaster L. 2012. Comparative real-time analysis of Saccharomyces

cerevisiae cell viability, injury and death induced by ultrasound (20 kHz) and heat for the

application of hurdle technology. Food Res Int 47(2):134-9.

Zafra-Rojas QY, Cruz-Cansino N, Ramírez-Moreno E, Delgado-Olivares L, Villanueva-Sánchez

J, Alanís-García E. 2013. Effects of ultrasound treatment in purple cactus pear (Opuntia

ficus-indica) juice. Ultrason Sonochem 20(5):1283-8.

Zhou L, Wang Y, Hu X, Wu J, Liao X. 2009. Effect of high pressure carbon dioxide on the quality

of carrot juice. Innovative Food Sci Emerging Technol 10(3): 321-7.

Zou Y, Jiang A. 2016. Effect of ultrasound treatment on quality and microbial load of carrot juice.

Food Sci Technol (Campinas) 36(1):111-5.

132

CHAPTER FOUR

ON THE INACTIVATION OF Listeria innocua IN CARROT JUICE BY COMBINING

ULTRASOUND, HEAT, AND NISIN

Abstract

This study investigated the effectiveness of ultrasound combined with thermal treatments and with

or without nisin (a natural antimicrobial peptide) in inactivating L. innocua as surrogate for L.

monocytogenes at non-isothermal conditions. Carrot juice (pre-pasteurized) was inoculated with

L. innocua and treated with ultrasound. Ultrasound amplitude, nisin level, and processing

temperature had a significant synergistic effect on the inactivation of L. innocua. When ultrasound

is combined with mild temperature and nisin, the microbial load can be effectively reduced in a

shorter time than with thermal or ultrasound processes used alone. For instance, we observed that

the application of nisin (25 ppm) at 20°C and processing for 5 min at 100% amplitude (400 W)

resulted in a 2.7 log reduction, while the same ultrasound and temperature conditions with 50 ppm

of nisin resulted in a 3.5 log reduction. This study reveals 5 conditions to achieve at least a 5 log

reduction, and further analyses performed after treatment with these conditions reveal no change

in pH, and in all but one condition, no significant change in TSS. There was increase in the

carotenoid content as well as significant differences in darkness, color, hue angle, and chroma for

all processed samples. This combination of low heat, a natural antimicrobial agent and ultrasound,

can contribute to reducing the energy required for processing, while increasing throughput. The

quality of the juice is retained as treatment is performed at milder temperatures and over a shorter

time compared to conventional thermal processing.

133

1. Introduction

Recent consumer demands for minimally processed and clean label food products has led to the

development of various alternative approaches to food processing. Ultrasound, pulsed electric

fields, high pressure processing, and microwave heating are some of the novel technologies that

have been widely studied over the last few decades, and have received special attention to process

nutritious beverages such as carrot juice.

Ultrasound is a clean, nonthermal technology recognized as a promising technology for the

pasteurization of fruit juices without affecting quality or nutritional attributes (Valero et al., 2007;

Bhat et al., 2011). Ultrasound technology has seen growing interest in the fruit and vegetable juice

processing industry due to many benefits, including low energy consumption, retention of nutrients

during processing, and fast processing time (Chemat and Khan 2011). The basic principle of

ultrasound in microbial inactivation is cavitation. When ultrasound is passed to the liquid medium,

bubbles are generated; these bubbles grow with time and finally collapse, resulting in localized

high pressure, which ultimately inactivates the microorganisms. Thermal effects and shock waves

due to bubble implosion, free radical formation, and mechanical stresses due to microstreaming

all contribute to microbial inactivation via ultrasound (Valdramidis et al., 2010).

Ultrasound alone has not been found to meet the FDA’s requirement for juice

pasteurization, i.e., a 5 log reduction in the viable microbial population (Piyasena et al., 2003). But

when ultrasound is used in combination with a mild temperature increase, synergy has been

observed (Wordon et al., 2012; Sango et al., 2014)). The process can be rendered energy-efficient,

and cost-effective by combining ultrasound and mild heating with natural antimicrobials such as

nisin. For example, when ultrasound is combined with nisin, a lower processing temperature can

134

be used to achieve the same value of lethality observed in conventional thermal processes

(Abdullah and Chin, 2014). Furthermore, the quality of the product can be retained as the

processing time is shorter and the temperature required is much lower than conventional thermal

processes. Many studies have shown that incorporation of natural and synthetic antimicrobial

agents can improve results in combination with ultrasound processing. Rodgers and Ryser (2004)

observed 5 log reduction of E. coli O157:H7 and L. monocytogenes in apple cider by combining

ultrasound treatment (44 to 48 Hz) with sodium hypochlorite (100 ppm) and copper ion water (1

ppm). Not many studies have been reported on the combination of ultrasound, heat, and nisin.

Carrot juice is a nutritious beverage rich in beta carotene and polyphenols (Sharma et al.,

2012). This product has low acid (pH>4.5) and is susceptible to microbial spoilage. The application

of high temperatures to preserve this juice negatively affects its nutritional and sensory attributes

of the juice. Ultrasound processing is one potential technology to process this juice without loss

of its desirable attributes.

Listeria monocytogenes is a heat-resistant food-borne pathogen, which can grows rapidly

under a wide range of pH and temperature conditions (Bermúdez-Aguirre et al., 2011; Gastélum

et al., 2012; Abdullah and Chin, 2014). This microorganism can contaminate a wide variety of

food products. L. innocua is one of the commonly used non-pathogenic surrogates for the pathogen

L. monocytogenes (Francis and O’Beirne, 1998; Buzrul and Alpas, 2004). It is generally

considered a target microorganism for pasteurization as it is highly heat resistant and can also grow

at refrigeration temperatures and at wide range of pHs.

The objective of this study was to investigate the potential of incorporating nisin during

ultrasound processing of carrot juice at different temperatures, and to study the synergism between

135

ultrasound, moderate temperature increases, and nisin during processing. The impact of these

processes on pH, color, TSS and carotenoid content of processed juice were also part of this study.

2. Materials and Methods

2.1 Juice preparation

Pre-pasteurized commercially available carrot juice (Bolthouse Farms, Bakersfield, CA) was

purchased from the local supermarket for initial testing. The pre-pasteurized juice was used for

microbiological tests to avoid interactions between inoculated microorganisms and natural

microflora. The juice had a pH of 6.4 and TSS of 8˚Brix. For subsequent testing (quality

evaluation), fresh carrot juice was prepared in the lab using a domestic juice extractor using carrots

purchased from the local supermarket; fresh juice was used to better evaluate the effect of selected

process on the base attributes. The fresh juice had a pH of 6.8 and TSS of 8.4°Brix.

2.2 Culture Preparation

A freeze-dried culture of Listeria innocua (ATCC 8456TM) was purchased; the dry culture was

activated in tryptic soy broth with 0.6% yeast extract, agitating at 215 rpm at 37˚C for 7 h. Cells

in the early stationary phase were harvested, mixed with 20% glycerol in water, and stored at -

18˚C. During the experiment, frozen stock culture was thawed and activated in tryptic soy broth

with 0.6% yeast extract.

2.3 Ultrasound Treatment

A carrot juice sample (250 mL) was placed in a 500-mL double layer jacketed glass beaker

connected to a water bath (VWR Scientific, Model 1166, Miles, IL) to control the temperature.

136

The ultrasonic probe was immersed in the juice 2 cm below the surface. Ultrasound treatment was

performed using an ultrasonic generator of 400 W at a constant frequency of 24 kHz using

amplitude levels of 60, 80, and 100% (72, 96, 120 µm) for a duration of 0 to 5 minutes. Acoustic

energy densities (AED) for each amplitude were 0.28, 0.37, and 0.48 W/mL respectively. The

sample was first heated with ultrasound and water bath to bring it to the target temperature. The

come-up time was about 2 minutes. After that, the juice sample was inoculated with previously

activated L. innocua (6 log), the water bath was turned off, and ultrasound was left on. The

temperature was monitored during processing using a k-type thermocouple (Omega Engineering,

Inc., Stamford, CT). A 1 mL aliquot was taken every minute for microbiological analysis.

Table 11. Final temperature of the juice after ultrasound processing for 5 min at different amplitudes

Amplitude

Initial Temperature

(°C)

Final Temperature (°C)

after 5 min

72 µm

20 38.6±0.9

35 39.4±0.7

50 60.6±1.5

96 µm

20 44.0±0.4

35 53.0±2.8

50 64.3±0.8

120 µm

20 50.7±1.4

35 59.6±1.0

50 68.8±1.0

2.4 Survivor Analysis

Two serial dilutions of the juice were performed in 0.1% sterile peptone water. One milliliter of

decimal dilutions of processed juice was pipetted into Petri dishes followed by addition of about

137

20 mL of tryptic soy agar with 0.6% yeast extract agar (45°C). The plates were incubated for 24

hours at 37°C, after which colonies were counted manually and expressed as CFU/mL.

2.5 Energy Consumption

Energy consumption during the treatment was calculated using the time, temperature, data

(temperature rise during processing). The calorimetric method was used to quantify the work done

(Watt) (Eq. 1). The specific heat of the carrot juice was taken as 3.77 kJ/kg°C as described by Raso

et al. 1999,

𝑄 = 𝑚 ∙ 𝐶𝑝 ∙𝑑𝑇

𝑑𝑡, (1)

where Q is the work done in watts, m is the mass of juice (kg), Cp is the specific heat of juice

(kJ/KgºC), and dT/dt is the change in temperature with respect to time (min).

Acoustic energy density (AED) was calculated by dividing the work done (Q) by the

volume of sample (Eq. 2),

𝐴𝐸𝐷 =𝑄

𝑉, (2)

where V is the volume of juice in mL.

Ultrasonic intensity was calculated by dividing the work done by area of the sonotrode (Eq.

3).

𝑈𝐼 =𝑄

𝐴, (3)

where A is area of the sonotrode (m2)

138

2.6 Optimum Processing Conditions and Quality Evaluation

Selection of the optimum processing conditions was based on target microbial load reduction (5

log), the lowest level of nisin, lowest amplitude, and shortest processing time. Selected processing

conditions were evaluated based on their effects on quality attributes of carrot juice. Processed and

fresh (control) juice were analyzed for pH, total soluble solids, color, and carotenoid content. Total

aerobic mesophilic bacteria were also quantified in fresh and processed juice.

2.6.1 Total Aerobic Mesophilic Bacteria

To quantify the total number of aerobic mesophilic bacteria in fresh and processed juice samples,

serial dilutions of the juice were made with 0.1% peptone water. Samples were pour-plated in Plate

Count Agar (Difco, Becton, Dickinson and Co., Sparks, MD) and incubated at 37°C for 48 h;

mesophiles were counted manually and expressed as log (CFU/mL).

2.6.2 pH, TSS, and color

The pH of the juices was determined using a pH meter (Orio 4-star, Thermo Fisher Scientific,

Waltham, MA, USA) at 21 ºC. Color parameters (Lightness, L*; redness, a*; and yellowness, b*)

of the juices were measured using a colorimeter (Konica Minolta, CM-5, New Jersey, USA). Total

soluble solid (TSS) of the juice was determined using a hand-held refractometer (Atago No. 480,

Atago Inc. Tokyo, Japan) at 21°C.

2.6.3 Total carotenoid content

Total carotenoid content of juices was analyzed using the method described by Liao et al. (2007).

In brief, 25 mL of juice was mixed with 80 mL hexane/acetone mixture (1:1, v/v) in a separating

139

funnel. After standing for a few minutes, the organic phase was separated from the aqueous phase

and collected. Absorbance was measured at 450 nm using a spectrophotometer (Spectronic 20

Genesys, Spectronic Instruments Inc., Rochester, NY, USA). The total carotenoid content was

determined using the standard curve of β-carotene and the results were expressed as mg β-carotene

per liter of sample.

3. Results and Discussion

3.1 Effect of Ultrasound on Listeria Inactivation

Treatment at room temperature (20°C) with no nisin resulted in a less than 1 log reduction in 5

min at all amplitude levels, well below the 5 log reduction required by the FDA for juice

pasteurization (Figure 20). When the initial product temperature was raised to 35°C there was still

less than a 1 log reduction at the 60% amplitude level, but at 80% and 100% amplitude levels,

there was a ~2 log reduction after 5 min of treatment. In the present study, the processing time was

short (<5 min); a higher level of inactivation could be achieved when processing time is increased.

Bauman et al. (2005) studied the effect of temperature on L. monocytogenes inactivation by

ultrasound in apple cider; the authors found a 1.6 log reduction from processing at a constant

temperature of 20°C for 20 min. Even though the processing time was 20 min, relatively long

compared to the 5 min of the present study, the reduction was still low. However in Bauman’s

work, the temperature was held constant whereas, in the present study, processing was conducted

under non-isothermal conditions. The 20°C treatment resulted in a final product temperature of

~51°C at 100% amplitude, leading to a 2 log reduction in only 5 min. Inactivation of

microorganisms by ultrasound depends on many factors; amplitude of the ultrasonic wave,

processing temperature, acoustic energy density, and ultrasonic intensity are some of the most

140

critical. In addition, the type of food product and type of microorganism targeted are also

determining factors in ultrasound processing.

Figure 20. Effect of ultrasound amplitude on inactivation of L. innocua in carrot juice at 20°C (left) and

35°C (right).

In the present study, different amplitude level had a significant difference (p>0.05) effect

on microbial inactivation. At high amplitudes, there was a significant rise in temperature, as well

as high acoustic energy density (AED) and ultrasonic intensity. The AED, ultrasonic intensity, and

rise in temperature all played a significant role in the higher level of inactivation with the increase

in amplitude. Comparing the effect of amplitude at room temperature (20°C) and a mild

temperature (35°C) after 4 minutes of processing, the difference between amplitude levels were

clearly visible (Figure 23, right). At room temperature, 60% amplitude and 80% amplitude there

were not significantly (p>0.05) different; similarly, no significant difference was observed

between these two amplitude at 35°C until three min. For both temperatures, there appear to be

shoulders in the inactivation curve for all the measured amplitudes: a shoulder was observed at 4

min for the treatment at 20°C, and at three min for the treatment at 35°C. No shoulders were

-1

-0.8

-0.6

-0.4

-0.2

0

0 2 4 6

Log

(N

t/N

0)

Time (min)

60% Amplitude

80% Amplitude

100% Amplitude

-2.5

-2

-1.5

-1

-0.5

0

0 2 4 6

Log

(N

t/N

0)

Time (min)

60% Amplitude

80% Amplitude

100% Amplitude

141

observed in the case of treatment at 50°C (Figure 24). The presence of the shoulder indicates that

bacteria were not affected by the treatment prior to that point. As already demonstrated and stated

by Bermúdez-Aguirre et al. (2011), the main phenomena happening during ultrasound treatment

responsible for microbial death are the cavitation formation and the cavitation explosion.

Ultrasound generates local cavitation characterized by a higher temperature and pressure than the

surrounding environment mainly after bubble collapse; in addition, cavitation increase the

turbulence of the solution. All these changes in physical environment can affect bacteria; however,

the impact depends on different factors including treatment time, the amplitude of ultrasound, and

the type of microorganism. Hence, the weak effectiveness of the previous treatments may be due

to the bacterial strain. In fact, L. innocua, as a gram-positive bacterium, is less sensitive to pressure

increases than gram-negative bacteria (Shiu et al., 2001); the elevated pressure in the cavitations

may not be high enough to cause irreversible damage to the bacterial cell wall. Bacteria can recover

from injuries by synthesizing new components; this may explain the bacterial growth observed

regardless the ultrasound amplitude after treatment was performed at 20 or 35°C without nisin.

This lag phase also may exist if the microbe repairs itself, as death occurs only when the rate of

destruction exceeds the rate of re-synthesis (Mossel et al., 1995).

3.2 Combined Effect of Ultrasound and Temperature on Listeria Inactivation

A synergistic interaction was observed between ultrasound and mild temperatures (35 and 50°C)

on Listeria inactivation. An increase in inactivation level was observed with increasing amplitude

levels, and or increasing temperature (Figure 21). After 35°C and 5 min of treatment, there was

around a 1 log reduction when using 80% amplitude, and around a 2 log reduction for 100%

amplitude. When the temperature was increased to 50°C, a 3.5 log reduction was achieved in 5

142

min at 60% amplitude; at 80% and 100% amplitude, microbial counts were below the detectable

limit (>6 log reduction) in 4 min and 3 min, respectively. Experiments performed by Ordoñez et

al. 1987, revealed a synergistic effect between ultrasound and heat on the inactivation of

Staphylococcus aureus after treatment performed between 50.3°C and 56.7°C at 20 kHz, 150 W

for 3 to 7 minutes. In the present study, at a 60% amplitude level, there was less than a 1 log

reduction at 20 and 35°C, and no major change was observed upon raising the temperature to 50°C;

a sigmoidal inactivation curve was observed with low amplitude/high temperature combinations.

This may be due to the homeostasis of bacteria: during the first 2 min of treatment at 60%

amplitude and 50°C, bacteria were affected by the environmental change; after that time, they may

adapt their behavior to survive, and since the treatment is not intense enough to cause irreversible

damage, they recover. These processes may explain why there was same level of inactivation after

3 or 4 min of treatment; nevertheless, with treatment times over 4 min, greater inactivation was

observed, potentially because the bacteria are injured beyond their capacity for repair.

When combining ultrasound and heat, there is an upper limit of temperature that is effective

during processing. Temperature above this critical limit has no additional antimicrobial effect

beyond that of thermal treatment used alone. This is due to a cushioning effect, and the reduction

in cavitation effects at high temperature (Ugarte-Romero et al., 2007). The upper limit of

temperature for a given process depends upon the type of food product, and is mainly influenced

by rheological characteristics, density, and surface tension.

143

Figure 21. Effect of temperature at different amplitude levels on the inactivation of L. innocua in carrot

juice (Data are mean of three replicates)

3.3 Combined Effect of Ultrasound, Temperature, and Nisin on Listeria Inactivation

At room temperature, interaction between ultrasound and nisin was effective only at high

amplitude (Figure 22). Even at 100% amplitude, ultrasound treatment at room temperature for 5

min did not meet the FDA requirements for juice pasteurization (>5 log reduction). Addition of

-5

-4

-3

-2

-1

0

0 2 4 6L

og

(N

t/N

0)

Time (min)

60% Amp

20°C 35°C 50°C

-8

-6

-4

-2

0

0 2 4 6

Log

(N

t/N

0)

Time (min)

80% Amp

20°C 35°C 50°C

-8

-7

-6

-5

-4

-3

-2

-1

0

0 1 2 3 4 5 6

Log

(N

t/N

0)

Time (min)

100% Amp

20°C 35°C 50°C

144

nisin increases the effectiveness of ultrasound treatment, but the inactivation was still low (<4 log)

for the selected treatment time (Table 12). These results are in line with those described by Muñoz

et al. (2012), who found that nisin (2.5 mg/L) combined with ultrasound led to a 2.8 log (CFU/mL)

reduction, whereas ultrasound alone led to only a 1.1 log (CFU/mL) reduction in Listeria.

Table 12. Effect of nisin concentration on the inactivation of L. innocua at room temperature (20°C).

Nisin concentration L. innocua Log Reduction

60% Amp 80% Amp 100% Amp

0 ppm 0.23 ±0.29 0.29 ±0.75 0.89 ±0.47

25 ppm 1.08 ±0.91 1.81 ±0.62 2.69 ±0.97

50 ppm 1.40 ±0.24 2.54 ±0.40 3.58 ±0.15

(Data are mean of three replicates ± standard deviation)

Incorporation of nisin with mild temperature elevation during ultrasound treatment

significantly enhanced the inactivation efficiency of ultrasound (Figure 25). At 80% amplitude and

35°C there was around a 2 log reduction in 5 min. But when 25 ppm nisin was added, the

inactivation was >4 log. A further increase in temperature to 50°C led to a 5.5 log reduction in L.

innocua. At 100% amplitude and 50°C, complete inactivation was achieved in 3 min without nisin,

and in 1 min with 25 ppm nisin. Similarly, for 80% amplitude at 50°C, complete inactivation was

achieved at 4 min without nisin, 2 min with 25 ppm nisin, and 1 min with 50 ppm nisin. Ultrasound,

temperature, and nisin act synergistically to affect bacteria. All of these treatments affect the

bacteria cell wall or membrane; in fact, according to Lubelski et al. (2008), nisin acts by creating

pores in the membrane of cells and inhibiting cell wall synthesis. Nisin can easily affect the

membrane of gram-positive L. innocua. Many biological reactions are affected by temperature,

145

including membrane fluidity, which may help nisin reach the cytoplasm. In addition, the impact

of ultrasound on the cell membrane may enhance the action of nisin.

In the present study, a combination of ultrasound with mild temperature and nisin reduced

the treatment time and amplitude by 20 min and more than 66%, respectively; these reductions aid

in retaining the quality attributes of juice. These reductions also render the process economically

desirable; amplitude reduction results in a reduction in energy consumption during processing

(Table 16).

-7

-6

-5

-4

-3

-2

-1

0

0 1 2 3 4 5

Log

(N

t/N

0)

Time (min)

80% Amplitude, 35°C

0 ppm 25 ppm 50 ppm

-8

-7

-6

-5

-4

-3

-2

-1

0

0 1 2 3 4 5

Log

(N

t/N

0)

Time (min)


0 ppm 25 ppm 50 ppm

146

Figure 22. Effect of different nisin concentration on inactivation of L. innocua at 35°C and 50°C

and at 80 and 100% amplitude level. Data are mean of three replicates

3.4 Energy Consumption during Processing

Temperature rise during processing was taken into account for energy use calculations. The

increase in temperature was dependent on the amplitude: at 60% amplitude, there was an increase

of about 15°C, and at 80 and 100% amplitude there was a rise of approximately 20 and 25°C,

respectively (Figure 23).

-8

-7

-6

-5

-4

-3

-2

-1

0

0 2 4 6L

og

(N

t/N

0)

Time (min)


0 ppm 25 ppm 50 ppm

-8

-7

-6

-5

-4

-3

-2

-1

0

0 1 2 3 4

Log

(N

t/N

0)

Time (min)


0 ppm 25 ppm 50 ppm

147

Figure 23. Temperature rise during processing at different amplitudes. Error bar represent the standard

deviation of three replicates

Acoustic energy density (AED) and ultrasonic intensity (UI) for each amplitude are

presented in Table 13. AED and UI were independent on the initial processing temperature, but

directly depend on the amplitude applied.

Table 13. Acoustic energy density and ultrasonic intensity at different amplitude levels (Values are the

average of three replicates ± standard deviation).

Amplitude

Power

Consumed (W)

Acoustic Energy Density (AED)

W/mL

Ultrasonic Intensity (UI)

W/cm2

60% 70.37±7.45 0.28±0.029 18.51±0.87

80% 93.10±5.65 0.37±0.022 24.49±1.48

100% 121.74±9.42 0.48±0.037 32.03±2.48

0

10

20

30

40

50

60

0 1 2 3 4 5 6

Tem

per

atu

re (°C

)

Time (min)

60% 80% 100%

148

As expected, there was increase in power with the increase in amplitude, and as a result the

AED and the UI also increase. There was a 73% power increase between the experiment performed

at 60% and that performed at 100%. Mantas et al. (2000) found a 128% increase in delivered power

at 100 kPa after processing at the amplitude level of 62 μm and 117 μm, where the delivered power

was 17.9 W and 40.9 W, respectively.

3.5 Selection of Optimum Processing Conditions

Five different processing conditions were selected with equivalent levels (5 log) of L. innocua

inactivation. No conditions at room temperature (20°C) were chosen, as none granted a 5 log

reduction. Addition of 25 ppm nisin, keeping amplitude and temperature constant, caused a ~66%

reduction in processing time. Similarly, keeping temperature and nisin level constant, even

treatments with a 20% reduction in amplitude could achieve the same level of microbial

inactivation. An 80% reduction in processing time was possible when the temperature was raised

from 35 to 50°C, keeping all other factors constant. Again, a 20% reduction in amplitude was

possible at 35°C with treatment time of 5 min when nisin concentration was increased from 25 to

50 ppm. These results demonstrate that a significant degree of reduction in both processing time

and amplitude level is possible with the incorporation of mild heating and nisin during ultrasound

processing.

Table 14. Processing conditions that results in at least a 5 log reduction of L. innocua

Condition Amplitude Temperature (°C) Nisin (ppm) Time (min)

a 80% Ti: 35, ΔT: 18 50 5

b 100% Ti: 35, ΔT: 24 25 5

c 60% Ti: 50, ΔT: 5 25 2

149

d 80% Ti: 50, ΔT: 12 0 4

e 100% Ti: 50, ΔT: 14 0 3

Ti is the initial temperature of the product before ultrasound treatment starts and ΔT is the

temperature difference between final temperature of the processed product and the initial

temperature.

3.6 Effect of Processes on Total Aerobic Mesophiles

The initial load of aerobic mesophilic bacteria in carrot juice was between 4 to 5 log. All the

selected treatments reduced the load of such bacteria to below 2 log. There was no significant

difference (p>0.05) between selected different equivalent processing conditions on the inactivation

of mesophiles (Figure 24). In a study conducted by Gómez-López et al. (2010) on the inactivation

of aerobic mesophilic bacteria in orange juice, authors observed 1.38 log reduction by using

ultrasound treatment (20 kHz, 500 W, 8 min, 89.25 µm, and 10°C). Although direct comparison

on the inactivation is not possible as there are many factors that affects the inactivation of

microorganisms, it can be said that the result from present study are in line with the findings of

Gómez-López. This study was focused on mild-pasteurization, hence it is expected to have some

level of natural flora in the treated juice. Complete inactivation of naturally occurring aerobic

mesophiles could be achieved when the intensity of the treatment is increased, for example the

processing at longer than 2 min.

150

Figure 24. Inactivation of mesophiles in carrot juice by selected combination of ultrasound treatment. No

significant difference (p>0.05) was found between selected equivalent processes on the inactivation of

mesophiles; Control indicates fresh juice without any treatments (a, b, c, d, e as shown in Table 14). Data

are mean of three replicates

3.7 Evaluation of Effects on Quality Attributes

3.7.1 pH, TSS, and Total Carotenoid Content

The selected processing conditions were also evaluated for their effects on the quality attributes of

treated fresh carrot juice. Changes in pH, TSS, and carotenoid content before and after treatment

are reported in Table 15. Slight changes in pH were observed after processing, but no significant

difference (p>0.05) in pH was noted between control and processed samples. There were changes

in the total soluble solids (TSS) content of the juice after treatment; the changes were not

significant for treatment conditions a, d, and e, while a significant increase in TSS was found for

conditions b and c. Nevertheless, results for samples treated with condition b are not significantly

different from the results for treatments a and d. The results found after treatment ‘c’ is also

significantly higher to the other processed sample; this particular treatment (60% amplitude, 50°C,

0

1

2

3

4

5

6

Control a b c d e

Log

(C

FU

)/m

L

151

25 ppm of nisin, 2 min) may affect TSS content. This may be due to the fact this treatment was

performed only for 2 minutes, while other treatments, in this and other studies, were performed

over longer periods. In the case of treatment e, which involved no nisin and the highest temperature

and amplitude, a slight decrease in °Brix was observed relative to control, although the change

was non-significant (p>0.05).

An increase in total carotenoid content relative to the control was observed in all the

processed samples. Significant increases were found only in processing conditions a and d; this

could be due to higher extraction of carotenoids after US treatment. Regardless, 100% retention of

carotenoid content of the juice was observed for all selected processing conditions.

Santhirasegaram et al. (2013), also found a significant increase in the carotenoid content of

Chokanan mango juice after ultrasound treatment performed at powers ranging from 68-75 W over

15 minutes. The same authors stated that this increase may be due to the ultrasound inactivating

enzymes which normally degrade carotenoids. Another explanation may be cell damage and an

increase in mass transport rate due to cavitation generated by ultrasound. These factors may allow

carotenoids to more readily spread from cells to the environment. This extraction may be favored

around 96 µm because cells must sustain enough damage to allow a high rate of extraction and

convection. In any case, the amount of carotenoids present in juice should not be affected by these

treatment conditions. Higher amplitudes in the range of 120 µm may affect carotenoids because of

the shear stress generated by cavitation collapse. This result demonstrates that combination of

ultrasound, temperature, and nisin not only inactivates microorganisms, rendering the product safe,

but also retains the nutrients present in the untreated juice.

152

Table 15. Change in pH, TSS, and carotenoid content of juice before and after processing under different

treatment conditions

Condition pH TSS (°Bx) Carotenoid (mg/L)

Control 6.20±0.04ab 9.27±0.06c 512.75±0.52c

a 6.34±0.12a 9.37±0.06bc 518.21±2.28ab

b 6.17±0.06b 9.73±0.15b 513.96±1.38c

c 6.31±0.01ab 10.20±0.20a 515.48±0.52bc

d 6.28±0.04ab 9.37±0.25bc 519.12±1.04a

e 6.31±0.02ab 9.20±0.10c 514.57±0.52c

(Values are the average of three replicates ± standard deviation. Different letter in the same column

represent that data are significantly different (p<0.05))

3.7.2 Color Parameters

There was a significant (p<0.05) reduction in the lightness of juice after treatment under all tested

conditions. This may be related to the increase in free carotenoid content discussed earlier, as β-

carotenoid is the main component responsible for the color of carrot juice. It follows logically that

an increase in the amount of free carotenoid would lead to an increase in the darkness of the

solution due to light absorption by free carotenoids. Similarly, there were significant reductions in

hue angle and chroma under all processing conditions. According to the description of chroma and

the hue angle given by McGuire (1992), a reduction in chroma means that color appears more

faded, less saturated. Since hue angle is defined as the angle between the hypotenuse and the a*

axis, a reduction in hue angle means here that the color is shifting towards blue, confirmed by the

corresponding observed decrease in the b* value. These changes may also be due to the rise in free

carotenoid.

153

In the measurements of color difference (ΔE), it was observed that all values ranged from

3.81 to 7.02. Thus, according to the rank established by Cserhalmi et al. (2006), the color difference

can be “noticeable” (3.0 to 6.0) or significant (6.0 to 12.0). The maximum change in color after

treatment was observed after treatment d, i.e., processing for 4 min at 80% amplitude, 50°C initial

temperature, and 50 ppm nisin. The combination of high temperature, mild amplitude, and longer

processing time may have caused this change in ΔE; samples subjected to condition d also showed

the highest levels of carotenoid after treatment, which may explain its greatest difference in color

relative to control.

Table 16. Change in Lightness, Hue angle, Chroma, and ΔE of the juice before and after processing with

different treatment conditions

Condition Lightness (L*) Hue angle (h*) Chroma (C*) ΔE

Control 9.75 ±0.20a 0.49±0.01a 35.58±0.31a -

a 7.81±0.16b 0.42±0.01b 32.53±0.18b 4.27±0.21c

b 7.11±0.07c 0.39±0.00c 31.26±0.16c 5.90±0.54b

c 7.67±0.07b 0.41±0.00b 32.38±0.11b 4.55±0.36c

d 6.51+0.05d 0.38±0.00d 29.81+0.08e 7.49±0.34a

e 6.99±0.05c 0.39±0.00c 30.86±0.12d 6.28±0.53b

(Values are the average of three replicates ± standard deviation. Different letter in the same column

represent that data are significantly different (p<0.05))

4. Conclusions

The simultaneous application of ultrasound, mild temperature and nisin for the processing of carrot

juice was found to be effective. Synergism was observed in these combinations for the inactivation

of L. innocua; in fact, 5 treatment conditions (a: 80% amplitude, 35°C, 50 ppm for 5 min; b: 100%,

154

35°C, 25 ppm for 5 min; c: 60%, 50°C, 25 ppm for 2 min; d: 80%, 50°C, 0 ppm for 4 min; and e:

100%, 50°C, 0 ppm for 3 min) were found to achieve at least 5 log reductions of Listeria innocua

using moderate temperature and amplitude. These combinations have great potential in the

production of minimally processed fresh fruit and vegetable juices. For all these conditions, we

find an increase in the carotenoid content, especially for the sample treated under condition c (80%

amplitude, 50°C, and 0 ppm nisin for 4 minutes). Since this treatment was performed only at 96

μm, it did not deliver the most energy, with an acoustic energy density of only 0.37 W/mL and an

ultrasonic intensity of 24.49 W/cm2. There was an increase in darkness for all treated samples, as

well as significant change in color, hue angle, and chroma, but no change in pH after processing.

The processing approach developed in this study will be beneficial for the industrial processing of

carrot juice and other vegetable juices without using very high temperature or chemical

preservatives. The proposed protocols will also help in the development of clean label products.

155

References

Abdullah, N., & Chin, N. L. (2014). Application of thermosonication treatment in processing and

production of high quality and safe-to-drink fruit juices. Agriculture and Agricultural

Science Procedia, 2, 320-327.

Bermúdez-Aguirre, D., Mobbs, T., & Barbosa-Cánovas, G. V. (2011). Ultrasound applications in

food processing. In Ultrasound Technologies for Food and Bioprocessing (pp. 65-105).

Springer New York.

Bhat, R., Kamaruddin, N. S. B. C., Min-Tze, L., & Karim, A. A. (2011). Sonication improves

kasturi lime (Citrus microcarpa) juice quality. Ultrasonics Sonochemistry, 18(6), 1295-

1300.

Buzrul, S., & Alpas, H. (2004). Modeling the synergistic effect of high pressure and heat on

inactivation kinetics of Listeria innocua: a preliminary study. FEMS Microbiology

Letters, 238(1), 29-36.

Chemat, F., & Khan, M. K. (2011). Applications of ultrasound in food technology: processing,

preservation and extraction. Ultrasonics Sonochemistry, 18(4), 813-835.

Cserhalmi, Z., Sass-Kiss, A., Tóth-Markus, M., & Lechner, N. (2006). Study of pulsed electric

field treated citrus juices. Innovative Food Science and Emerging Technologies, 7(1), 49-

54.

Francis, G. A., & O'Beirne, D. (1998). Effects of the indigenous microflora of minimally processed

lettuce on the survival and growth of Listeria innocua. International Journal of Food

Science and Technology, 33(5), 477-488.

156

Gastélum, G. G., Avila-Sosa, R., López-Malo, A., & Palou, E. (2012). Listeria innocua multi-

target inactivation by thermo-sonication and vanillin. Food and Bioprocess

Technology, 5(2), 665-671.

Gómez-López, V. M., Orsolani, L., Martínez-Yépez, A., & Tapia, M. S. (2010). Microbiological

and sensory quality of sonicated calcium-added orange juice. LWT-Food Science and

Technology, 43(5), 808-813.

Liao, H., Sun, Y., Ni, Y., Liao, X., Hu, X., Wu, J., & Chen, F. (2007). The effect of enzymatic

mash treatment, pressing, centrifugation, homogenization, deaeration, sterilization and

storage on carrot juice. Journal of Food Process Engineering, 30(4), 421-435.

Lubelski, J., Rink, R., Khusainov, R., Moll, G. N., & Kuipers, O. P. (2008). Biosynthesis,

immunity, regulation, mode of action and engineering of the model antibiotic

nisin. Cellular and Molecular Life Sciences, 65(3), 455-476.

Mantas, P., Pagán, R., & Raso, J. (2000). Predicting lethal effect of ultrasonic waves under pressure

treatments on Listeria monocytogenes ATCC 15313 by power measurements. Journal of

Food Science, 65(4), 663-667.

McGuire, R. G. (1992). Reporting of objective color measurements. HortScience, 27(12), 1254-

1255.

Mossel, D. A. A., Corry, J. E., Struijk, C. B., & Baird, R. M. (1995). Essentials of the microbiology

of foods: a textbook for advanced studies. John Wiley & Sons.

157

Muñoz, A., Palgan, I., Noci, F., Cronin, D. A., Morgan, D. J., Whyte, P., & Lyng, J. G. (2012).

Combinations of selected non-thermal technologies and antimicrobials for microbial

inactivation in a buffer system. Food Research International, 47(1), 100-105.

Ordoñez, J. A., Aguilera, M. A., Garcia, M. L., & Sanz, B. (1987). Effect of combined ultrasonic

and heat treatment (thermoultrasonication) on the survival of a strain of Staphylococcus

aureus. Journal of Dairy Research, 54(1), 61-67.



Raso, J., Manas, P., Pagan, R., & Sala, F. J. (1999). Influence of different factors on the output

power transferred into medium by ultrasound. Ultrasonics Sonochemistry, 5(4), 157-162.

Rodgers, S. L., & Ryser, E. T. (2004). Reduction of microbial pathogens during apple cider

production using sodium hypochlorite, copper ion, and sonication. Journal of Food

Protection, 67(4), 766-771.

Sango, D. M, Abela, D., McElhatton, A., & Valdramidis, V. P. (2014). Assisted ultrasound

applications for the production of safe foods. Journal of Applied Microbiology, 116(5),

1067-1083.

Santhirasegaram, V., Razali, Z., & Somasundram, C. (2013). Effects of thermal treatment and

sonication on quality attributes of Chokanan mango (Mangifera indica L.)

juice. Ultrasonics Sonochemistry, 20(5), 1276-1282.

158

Sharma, K. D., Karki, S., Thakur, N. S., & Attri, S. (2012). Chemical composition, functional

properties and processing of carrot—a review. Journal of Food Science and

Technology, 49(1), 22-32.

Shiu, C., Zhang, Z., & Thomas, C. R. (2001). A comparison of the mechanical properties of

different bacterial species. In Applied Microbiology (pp. 155-162). Springer Netherlands.

Valdramidis, V. P., Cullen, P. J., Tiwari, B. K., & O’Donnell, C. P. (2010). Quantitative modelling

approaches for ascorbic acid degradation and non-enzymatic browning of orange juice

during ultrasound processing. Journal of Food Engineering, 96(3), 449-454.

Valero, M., Recrosio, N., Saura, D., Muñoz, N., Martí, N., & Lizama, V. (2007). Effects of

ultrasonic treatments in orange juice processing. Journal of Food Engineering, 80(2), 509-

516.



and heat for the application of hurdle technology. Food Research International, 47(2),

134-139.

159

CHAPTER FIVE

INACTIVATION OF Listeria innocua AND Escherichia coli IN CARROT JUICE BY

COMBINING HIGH PRESSURE PROCESSING, NISIN, AND MILD

THERMAL TREATMENTS

Abstract

This study explores the effectiveness of High Pressure Processing (HPP) together with mild

thermal treatments and nisin on the inactivation of L. innocua and E. coli in carrot juice. Carrot

juice samples, with or without nisin (0-25-50 ppm), were inoculated with L. innocua or E. coli

(one at a time), and subjected to pressures of 200, 300, 400 or 500 MPa for 2 min at three different

temperatures, 20, 35, and 50°C.

Processing at 500 MPa at 20°C without nisin resulted in a 4 log reduction of L. innocua

and a 5 log reduction of E. coli, while the incorporation of 25 ppm of nisin at the same pressure

and temperature rendered 7 log reductions in both microorganisms. For the processing at 35°C,

300 MPa, 25 ppm nisin, there was more than 5 log reduction. Processing conditions that resulted

in at least 5 log reduction for both microorganisms had no significant (p>0.05) impact on the juice


There were synergisms among HPP, nisin, and mild temperature in the inactivation of L.

innocua and E. coli. Incorporation of nisin and processing at mild temperatures allow the use of

less intense HPP treatments, to pasteurize carrot juice making the process energy efficient and cost

effective. The combination of nisin, HPP, and mild temperatures could be useful in the commercial

pasteurization of carrot juice and other low-acid juices to attain safe, clean label, and high quality

products.

160

1. Introduction

Carrot juice is a highly nutritious beverage rich in beta carotene and antioxidants. But, it has a pH

in the range of 6.4-6.8, making it challenging to extend shelf life, as undesirable microorganisms

are very resistant at these pH values. Conventional thermal methods use relatively high

temperatures (70 to 90°C) with a holding time of 1 to 6 minutes. These high temperatures and long

holding times have been reported to cause undesirable quality and nutritional degradation in juices

(Plaza et al., 2006), as well as in degradation of heat-sensitive bioactive compounds. One

alternative to minimize such drawbacks is to use nonthermal food processing technologies which

have been developed in the last few decades. Food process engineers are constantly searching for

novel methods to inactivate undesirable microorganisms with little or no effect on the nutritional

and quality attributes of the food. High pressure processing (HPP), a nonthermal technology, is an

excellent alternative to process food. The process can inactivate undesirable microorganisms and

has minimal or no effect on the nutritional and quality attributes of foods.

The major governing factor in the inactivation of microorganisms in HPP is pressure;

however, some amount of heat can be generated during the process, which also contributes to

microbial inactivation. This technology has numerous advantages over conventional processes; for

example, HPP has no adverse effects on bioactive compounds of foods, as the pressure does not

alter covalent bonds. Furthermore, HPP is independent of the size and shape of food products, as

it is an isostatic phenomenon (Patras et al., 2009a). Food and beverages processed by HPP have

been shown to have fresh-like quality, flavor, taste, and nutrients (Dede et al., 2007; Oey et al.,

2008; Zhang et al., 2016).

Despite its many advantages, HPP also has some limitations. The process is carried out

either in batch or semi-continuous mode, and the high level of pressure and long holding time

161

result in lower throughput and higher cost of operation. To lower the cost of processing and

increase throughput, the processing pressure and holding time need to be reduced. One way to

efficiently reduce both factors is to combine high pressure with other hurdles such as moderate

temperature increases (<50°C) and natural antimicrobials (Bermúdez-Aguirre et al., 2016).

Nisin is a heat-stable bacteriocin produced from Lactococcus lactis and has been classified

in the Generally Recognized as Safe (GRAS) categories by US FDA and can be used in canned

food, dairy products and meat (de Arauz et al., 2009; Tong et al., 2014). Gram-positive bacteria

are sensitive to nisin (Brewer et al., 2002). But, studies have shown that nisin is less effective

toward gram-negative bacteria as compared to gram-positive (Helander & Mattila-Sandholm,

2000). The main mechanism of inactivation by nisin is its ability to form ion-permeable pores in

the cytoplasmic membranes of microbial cells (de Arauz et al., 2009; Smith et al., 2002). Nisin on

its own is less effective for inactivation of gram-negative microbes as compared to gram-positive

ones. In combination with HPP, though, it could be effective for gram-negative bacteria, as HPP

can cause pores in the outer cell membrane allowing nisin to reach and act on the cytoplasm

membrane, leading to lysis.

The objective of this work was to study the combined effect of HPP, nisin, and moderate

temperature increases on the inactivation of a representative gram-positive bacteria, L. innocua,

and a gram-negative bacteria, E. coli in carrot juice, selected to represent low acid foods (pH >

4.6). Such combinations have not been previously reported for carrot juice. The other objective of

this work was to investigate the effect of such combinations on the quality and stability of treated

carrot juice.

162


2.1 Culture preparation

E. coli (ATCC 11755TM) and L. innocua (ATCC 8456TM) were obtained from American Type

Culture Collection. The strains of L. innocua and E. coli were grown in 100 mL of sterile tryptic

soy broth (TSB) + 0.6% yeast extract (YE) and nutrient broth (NB) (Becton Dickinson, Sparks,

MD, USA), respectively, at 37°C under agitation at 225 rpm in a shaking water bath (Lab-Line,

model 3545, Melrose Park, IL, USA). The cells were harvested at the early stationary phase, and

the cultures frozen at -18°C in 20 % sterile glycerol until use. To activate the frozen culture, vials

with E. coli and L. innocua were defrosted and added to nutrient broth or TSB at room temperature

and shaken for 10 min.

2.2 Sample preparation

In the first part of the study, thermally pasteurized refrigerated carrot juice (Bolthouse Farms,

Bakersfield, CA, USA) (pH 6.4; soluble solids 11.7°Bx) without any preservatives were purchased

at a local grocery store (Pullman, WA, USA). A 100 mL aliquot of juice was aseptically dispensed

into a sterile Erlenmeyer flask (250 mL). Pure nisin-A (Handary S.A, Brussels, Belgium) was

added to the juice to achieve concentrations of 25 and 50 ppm. Juice without nisin was also used

for comparison to examine the effects of nisin addition. Twenty milliliters of the juice with- or

without nisin was placed in sterile Nylon/PE pouches (50 mL) for processing. Cultures of E. coli

and L. innocua (one at a time) were inoculated in the juice pouch to reach a final concentration of

7 log CFU/mL. Juice pouches with the microbial culture were sealed using a lab-scale sealer

163

(Impulse Heat Sealer 3T06, Midwest Pacific, Taiwan). HPP treatment was performed immediately

after sealing.

In the second part of this study, fresh carrot juice was prepared in the lab using a domestic

juicer (Hamilton Beach JE2200B, Black & Decker, MD, USA). Fresh juice was used to monitor

quality changes after high pressure treatment and during storage. Figure 25 shows the process flow

diagram for High Pressure Processing of nisin-incorporated carrot juice at mild temperatures.

164

Figure 25. Process flow diagram for High Pressure Processing of nisin-incorporated carrot juice at mild

temperatures

2.3 High Pressure Processing (HPP)

The juice pouches were processed using a HPP unit (Engineering Pressure Systems, Inc., Andover

MA, USA) located in the Washington State University Food Processing Pilot plant. A 5% Mobil

Hydrasol 78 water solution was used as the pressure-transmitting medium. Pressures of 200, 300,

400, and 500 MPa with a holding time of 2 min were used for treatment of the juices. The initial

Carrot Juice (Pasteurized, Bothhouse

farms)

Addition of nisin (0, 25, 50 ppm) at 20ºC

Innoculation of L. innocua or E. coli (7 log)

at 20ºC

Packaging of juice (Nylon/PE pouches) and

Sealing

High Pressure Processing (200-500 MPa, 20-50°C,

2 min)Microbiological analysis

Selection of processing conditions that render at least 5 log reduction of both microorganisms

Calculation of energy consumption during

processing

Application of selected processing conditions to

fresh carrot juice

Analyse the inactivation of naturally occuring

total aerobic mesophilic bacteria in fresh carrot

juice

Study changes in physicochemical

characterstics of the juice

Storage studies for 4 weeks at 4°C

(Quality evaluation)

165

temperature of the pressurizing medium was set to 20, 35 and 50°C. The compression heating

resulted in temperature rise of the processing fluid and juice (by up to 7, 10, 13, and 14°C at 200,

300, 400, and 500 MPa, respectively). Control samples (fresh juice) were held at atmospheric

pressure and room temperature (20°C) while processing the other samples.

2.4 Enumeration of viable E. coli and L. innocua

Microbiological analysis was conducted to study the inactivation of both microorganisms in carrot

juice after high pressure processing. Serial dilutions were prepared in triplicate with 0.1% (w/v)

peptone water (Becton, Dickinson and Company, Sparks, MD). One mL of decimal dilution of

sample was pipetted into Petri dishes, and 20 mL of tryptic soy agar plus yeast extract (for L.

innocua) or MacConkey agar (for E. coli) was poured to each plate. Plates were incubated at 37ºC

for 48 hours after being turned upside down. All tests were performed in triplicate; survivors were

counted manually and represented as colony forming unit (CFU)/mL.

2.5 Quality evaluation and storage studies

Physicochemical characteristics of fresh and processed carrot juice were evaluated for changes in

quality after incorporation of nisin, and by the application of high pressure at different

temperatures. Operating conditions that were successful in achieving 5 log reductions of selected

microorganisms were chosen for this purpose. Processed samples, along with an unprocessed

control samples were stored at 4°C for 4 weeks to monitor the growth of aerobic mesophilic

bacteria and changes in pH, color, soluble solids, and turbidity. All the samples were stored in the

same pouches that were used to process, pouches had a water vapor transmission rate of 6-7.5

g/m2.24 hr. and oxygen transmission rate of 50-70 cc/m2.24 hr.

166

2.5.1 Physicochemical Analysis

In this study, physicochemical quality attributes such as pH, color, turbidity, total soluble solids,

ascorbic acid, total carotenoid content, and total phenolic compounds were determined to evaluate

the juice sample before and after processing as well as during the storage period.

a. pH, Color, Turbidity, and Total Soluble Solids

The pH of the juices was evaluated using a digital pH meter (Orio 4-star, Thermo Fisher Scientific,

Waltham, MA, USA) at 20ºC. Hunter color parameters (Lightness, redness, and yellowness) of

the juice samples were measured using a colorimeter (Konica Minolta, CM-5, New Jersey, USA).

To measure turbidity, a sample was centrifuged (1500 RPM, 10 min) (MSE Centaur 2 centrifuge

(MSE UK Ltd., London, UK), the supernatant taken, and the absorbance at 660 nm measured using

a spectrophotometer (Spectronic 20 Genesys, Spectronic Instruments Inc., Rochester, NY) (Krop,

1974). The total soluble solids content of the juice was quantified using a hand-held refractometer

(Atago No. 480, Atago Inc. Tokyo, Japan) at 20°C.

b. Ascorbic Acid

The ascorbic acid level in fresh and processed juices was determined by redox titration method

using iodine and starch indicator, according to the method described by Ciancaglini et al. (2001),

with some modifications. A juice sample (20 mL) was pipetted into a 250-mL Erlenmeyer flask,

and 150 mL of distilled water and 1 mL of starch indicator were added. The sample solution was

titrated with a 5 mM iodine solution until a permanent green-black color was first observed.

167

c. Total Carotenoid Content

The juice before and after processing was analyzed spectrophotometrically according to the

method described by Liao et al. (2007) to determine total carotenoid content. Twenty milliliters of

juice was mixed with 20 mL of a hexane/acetone mixture (1:1, v/v) in a separating funnel and

shaken. After one minute, the organic phase separated from the aqueous phase was collected, and

the absorbance was measured at 450 nm using a spectrophotometer (Spectronic 20 Genesys.

Spectronic Instruments Inc., Rochester, NY, USA). The total carotenoid content was calculated

using a standard curve of beta-carotene with concentrations ranging from 2 to 10 mg/L. Results

were expressed as milligrams of β-carotene per liter of sample. All the analyses were conducted

in triplicates.

d. Total Soluble Phenolic Content

The method described by Derradji-Benmeziane et al. (2014) was used to determine total soluble

phenolic content. Juice sample (50 µL) was placed into a volumetric flask, and 2.5 mL of Folin-

Ciocalteu reagent (diluted 1/10) was added. Each sample was left to stand for 10 minutes at room

temperature, after which 2 mL of an aqueous solution of Na2CO3 (7.5%) was added, and the sample

kept for 30 min in the dark. Absorbance was determined at 760 nm, and phenolic compounds were

quantified using a standard prepared using Gallic acid. The results were expressed in milligrams

equivalent Gallic acid per liter of juice. All the analyses were conducted in triplicates.

168

2.6 Energy calculation

Since each process results in a combination of pressure and temperature increase, energy

consumed to raise the pressure and the temperature were calculated separately according to

equations (1) and (2) respectively (Silva et al., 2016):

𝐸 =1

2𝑉 × 𝛽 × (𝑃𝑓𝑖𝑛𝑎𝑙

2 − 𝑃𝑖𝑛𝑖𝑡𝑖𝑎𝑙2) − 𝑃𝑓𝑖𝑛𝑎𝑙 × 𝑉𝛼(𝑇𝑓𝑖𝑛𝑎𝑙 − 𝑇𝑖𝑛𝑖𝑡𝑖𝑎𝑙) + 𝜌 × 𝑉 × 𝐶𝑝 ×

𝑑𝑇

𝑑𝑡, (1)

𝑄 = 𝑉 × 𝜌 × 𝐶𝑝 × 𝑑𝑇, (2)

where ρ (kg/m3) is the density of the fluid, Cp (kJ/kg°C) the mass heat capacity of the fluid, dT the

temperature difference (in °C or K), V the volume of cylinder (m3), P (MPa) and T (ºC) the pressure

and the temperature in the vessel, respectively, α the expansivity (6.07E-4 K-1 or °C-1), β the

compressibility (4.6E-10 Pa-1) of water used as pressure transmitting fluid, and dT/dt the increase

in experimental temperature of the chamber during the HPP come-up time. Results for E were

expressed in kJ/L, obtained by dividing the total energy required by 2 L, the high-pressure vessel

capacity.

2.7 Statistical analysis

Data analysis was performed using Microsoft Excel 2016. The analysis of variance (ANOVA)

between different processing conditions was analyzed using Minitab 16 (Minitab, State College,

PA, USA). A p-value of 0.05 was chosen as the limit for significance.

169


This section highlights the significant findings on the inactivation of gram positive and gram-

negative microorganisms by HPP, nisin and mild thermal treatments. To that end, treatment impact

on microbial inactivation was investigated for HPP alone, HPP combined with nisin, HPP

combined with heat, and for all three parameters combined. Additionally, it presents the effects of

each selected combination on the physical and chemical properties of treated carrot juice, as well

as the energy consumption and the stability of the treated juice over time.

3.1 Effect of process on the inactivation of L. innocua and E. coli

Several tests were conducted with a different level of pressure, nisin, and temperature to assess the

effect of each process or of a combined process on bacterial inactivation. The results obtained was

used to determine the optimum combination of pressure, temperature, and nisin concentration to

obtain at least 5 log reduction of L. innocua and E. coli.

3.1.1 Effect of HPP

A steady reduction of both L. innocua and E. coli were found with an increase in processing

pressure, as shown in Figure 2, which clearly indicates that the application of HPP alone has a

significant impact on the inactivation of both microorganisms. This effect is more profound at

pressures higher than 400 MPa. Application of 200 MPa at 20°C reduced the L. innocua and E.

coli count by less than 1 log. Increasing the pressure to 300 MPa resulted in a 1 log reduction of

170

E. coli, but under the same conditions, L. innocua reduction was still less than 1 log. By increasing

the pressure level to 400 MPa, a 3 log reduction was achieved for both microorganisms.

Figure 26. Effect of increased pressure at room temperature (20°C) on reduction of L. innocua and E.

coli.

A further increase in pressure to 500 MPa rendered about 4 and 5 log reductions of L.

innocua and E. coli, respectively. These results show that L. innocua was more resistant to pressure

than E. coli, which is consistent with previous findings that gram-negative bacteria are more

susceptible to physical stress than gram-positive bacteria (Shiu et al., 2001). This difference in

sensitivity has been attributed to the complexity of the gram-negative cell membrane compared to

the gram-positive cell membrane (Murchie et al., 2005). In fact, these two categories of bacteria

have major differences in the structure of the cell membrane. Gram-negative bacterial cells have

an outer membrane composed of purines, lipopolysaccharides, and phospholipids. All of these

components may be kept together by weak forces such as hydrophobic and Van der Waals

0

1

2

3

4

5

6

200 300 400 500

Log

Red

uct

ion

Pressure (MPa)

L. innocua E. coli

171

interactions, which are affected by HPP. The outer membrane of gram negative cells also contains

proteins in an antiparallel β-strands conformation (Bos et al., 2007). Since HPP affects weak

bonds, therefore, it affects protein conformation like antiparallel β-strands, which may be another

reason for the higher sensitivity of gram-negative bacteria to high pressure. On the other hand,

gram-positive bacteria walls have peptidoglycan layers, held together by covalent bonds. HPP has

no significant effects on covalent bonds; hence, the stronger gram-positive bacteria are preserved

while gram-negative bacteria are damaged. Similar results were observed by Wuytack et al. (2002)

on the inactivation of gram positive and negative bacteria by high pressure.

Application of 500 MPa for 2 min alone did not reach the carrot juice pasteurization criteria

of this study (>5 log reduction). An increase in pressure beyond 500 MPa could be challenging to

implement in a food plant, as the increase involves higher costs for equipment and processing. An

increase in processing time is associated with lower productivity or throughput. Generally, a

processing time of 2 minutes or less is desired by fast-paced industry. Therefore, to make HPP

efficient in terms of equipment cost and productivity, a combination of HPP with certain hurdles

is a sound approach, especially for processing low-acid juices.

3.1.1 Study of combined effect

The results obtained from test that combines nisin with HPP and heat are summarized in Figure

27.

172

-8

-7

-6

-5

-4

-3

-2

-1

0

0 100 200 300 400 500L

og

(N

p/N

0)

Pressure (MPa)

L. innocua Inactivation at 20 °C

0 ppm 25 ppm 50 ppm

-8

-7

-6

-5

-4

-3

-2

-1

0

0 100 200 300 400 500

Log

(N

p/N

0)

Pressure (MPa)

L. innocua Inactivation at 35 °C

0 ppm 25 ppm 50 ppm

-9

-8

-7

-6

-5

-4

-3

-2

-1

0

0 100 200 300 400 500

Log

(N

p/N

0)

Pressure (MPa)

E. coli Inactivation at 20°C

0 ppm 25 ppm 50 ppm

-8

-7

-6

-5

-4

-3

-2

-1

0

0 100 200 300 400 500

Log

(N

p/N

0)

Pressure (MPa)

E. coli Inactivation at 35°C

0 ppm 25 ppm 50 ppm

173

Figure 27. The combined effect of HPP, nisin, and heat on microbial inactivation. Data represent the

mean of three replicate

a. Combined effect of HPP and nisin

The application of nisin without HPP treatments had a minimal impact on both L. innocua and E.

coli. In gram-negative bacteria, the permeation of nisin to the cytoplasmic membrane is protected

by a lipid layer on the outer membrane (Jack et al., 1995). Previous studies show that nisin was

effective only against gram-positive bacteria since it can quickly reach the cytoplasmic membrane

and has the capacity to permeabilize it and inhibit cell wall synthesis (Lubelski et al., 2008).

Results suggest a higher resistance of E. coli towards nisin over L. innocua, an observation in line

with those of McNamee et al. (2010). Nisin can easily reach the cell membrane of L. innocua but

cannot pass through the complex outer membrane of E. coli. Several studies have shown that nisin

alone has little or no effect on gram-negative bacteria like E. coli (Boziaris & Adams, 1999;

Helander & Matitila-Sandholm, 2000). In the present study, when nisin was combined with

pressure, there was synergism. This enhancement in the effect of nisin, when combined with

-8

-7

-6

-5

-4

-3

-2

-1

0

0 100 200 300 400 500 600

Lo

g (

Np

/N0)

Pressure (MPa)

L. innocua Inactivation

20°C 35°C 50°C

-8

-7

-6

-5

-4

-3

-2

-1

0

0 100 200 300 400 500 600

Lo

g (

Np

/N0)

Pressure (MPa)

E. coli Inactivation

20°C 35°C 50°C

174

pressure, may be due to partial rupture of the outer cell membrane by HPP, allowing nisin to pass

through that wall and reach the cytoplasmic membrane causing pores and lysis and inactivating

the microorganisms.

It has been reported that the binding of nisin to the cell membrane of gram-positive

microorganisms increases the susceptibility of microorganisms to high pressure treatments and

results in synergism (ter Steeg et al., 1999; Lee et al., 2003). The synergy between HPP and nisin

on the inactivation of microorganisms depends on factors such as treatment medium and

microorganisms type (Sokołowska et al., 2012).

Addition of nisin at 25 and 50 ppm did not impact the lethal effect at pressures of 200 or

300 MPa and at room temperature (20°C). The applied pressure levels might not be strong enough

to cause change in bacterial cell membrane thereby preventing the nisin to easily penetrate into the

cytoplasm. Increasing the pressure from 300 to 400 MPa with the same concentrations of nisin and

at the same temperature had a significant impact on the inactivation of both microorganisms. There

was a synergistic interaction between nisin and pressure to inactivate both tested microorganisms.

These results illustrate that the combination of HPP and nisin is an alternative which has potential

for the food and beverage processing industry.

b. Combined effect of HPP and heat

Combination of different pressures and mild temperatures significantly (p<0.05) reduced

microbial population, as can be seen in Figure 27. Microbial inactivation increased with increases

in pressure and/or temperature. At low pressure levels (200 and 300 MPa) the effect of raising the

temperature from 20°C to 35°C had less impact than the same increase at pressure above 400 MPa.

175

For the three levels of pressure, a significant increment in log reduction was observed when the

temperature was raised to 50°C. A pressure of 400 MPa at 35°C resulted in more than 6 log

reduction of both microorganisms, and when the temperature was raised to 50°C at this pressure,

bacterial inactivation was higher than 7 log (below the detectable limit). At 500 MPa, 35°C, 7 log

inactivation of both microorganisms was achieved. The same level of reduction of E. coli was

observed at 300 MPa and 50°C.

c. Combined Effect of HPP, nisin, and temperature

Figure 27 shows that the combined effect of HPP and nisin was further enhanced by a moderate

temperature elevation during processing, which is in line with many other studies combining high

pressure and nisin at or above room temperature (Hauben et al., 1996; Kalchayanand et al., 2004).

This enhancement may be explained by membrane fluidity changes due to heating. Hence, similar

effects of a rise in pressure and temperature on the membrane allows nisin to penetrate through it

more easily.

3.2 Effect of treatments on the quality of fresh carrot juice

The optimum processing conditions (Table 17) that led to at least 5 log reduction of both L. innocua

and E. coli were selected to further study the effects of treatment combinations on the quality

attributes of the juice.

176

Table 17. Optimum processing conditions that led to at least 5 log reduction of L. innocua and E. coli.

Combination ‘a’ is control or fresh untreated juice.

Combinations Pressure (MPa) Temperature (°C) Nisin (ppm)

a (Control) 0.1 (Atmospheric pressure) 20 0

b 200 50 50

c 300 35 25

d 400 35 0

e 500 20 0

3.3 Effect on pH, total soluble solids (TSS), and turbidity

There was a significant reduction (p<0.05) in pH after processing by all selected combinations of

pressure, nisin, and heat as compared to control (Table 18). Bonds between hydrogen and acidic

molecules are reversible and may be affected by HPP. Hence, an increase in pressure might lead

to release hydrogen ions from acids to the environment, leading to a decrease in pH.

Soluble solids are responsible for sensory properties of the juice; retention of them helps

preserve the sensory quality of fresh juices after processing. Significant decreases in total soluble

solids (TSS) were observed for samples b, c, and d, treated at temperatures above ambient. Sample

e, treated at 500 MPa, is the only sample that did not exhibit any significant decrease in TSS

relative to the control sample, and the only sample treated at room temperature. As discussed

earlier, a pressure of 500 MPa is high enough to cause membrane cell disruption. All these

observations may suggest that only the increase in temperature (and perhaps not the increase in

pressure) undergone by samples b, c and d may damage sugars present in the juice, leading to the

significant decrease in total soluble solids.

177

Slight decreases in turbidity values were observed in all of the treated samples compared

to the control. The turbidity value indicates the light-scattering properties of a solution; and

therefore depends on several factors like the particles’ size. Hence, the decrease in turbidity might

be explained by a reduction in the size of particles dispersed in the juice since pressure causes

rupture or breakage of fruit cells.

Table 18. Effect of combination of HPP, temperature, and nisin on pH, soluble solids, and turbidity.

Sample pH TSS (°Bx) Turbidity (Abs. at 660 nm)

a 6.60±0.01a 9.35±0.14a 2.32±0.02a

b 6.40±0.01c 8.25±0.05c 2.09±0.01b

c 6.42±0.01bc 8.20±0.10c 2.02±0.01b

d 6.44±0.01b 8.50±0.10bc 2.01±0.04b

e 6.41±0.01bc 9.05±0.05ab 2.04±0.03b

The values are mean ± standard deviation. The same letter (superscript) after standard deviation in

the column indicate no significant difference (p>0.05) between the samples.

3.4 Effect on color characteristics

There was no significant difference (p>0.05) in lightness (L*), Chroma (C*), and Hue angle (h*)

values between the control and samples processed by different combination of pressure, nisin and

temperature (Table 19).

178

Table 19. Effect of combinations of HPP, temperature, and nisin on the color characteristics of carrot

juice

Samples Lightness (L*) Chroma (C*) Hue Angle (h*)

a. Control 12.09±0.41a 38.63±0.95a 0.56±0.01a

b. 200 MPa, 50 ppm nisin, 50°C 10.86±0.62a 36.78±1.40a 0.53±0.02a

c. 300 MPa, 25 ppm nisin, 35°C 11.03±0.15a 36.68±0.34a 0.54±0.00a

d. 400 MPa, 0 ppm nisin, 35°C 10.66±0.67a 35.79±1.72a 0.53±0.02a

e. 500 MPa, 0 ppm nisin, 20°C 8.98±1.22a 33.30±2.52a 0.48±0.06a

The values are a mean ± standard deviation. The same letter (superscript) after standard deviation in

the column indicates no significant difference between the samples.

3.5 Effect on Bioactive compounds

Since carrot juice has several essential bioactive compounds, it is of interest to study the effect of

each selected treatment on those compounds. Hereby, changes on the total carotenoids, phenolic

compounds and ascorbic acid content were determined.

3.5.1 Total carotenoids

There was a slight but non-significant (p>0.05) increase in total carotenoid concentration in

samples treated by HPP at 300 MPa, 400 MPa, and 500 MPa, and a slight but also non-significant

(p>0.05) decrease for the sample treated at 200 MPa at 50°C (Table 20). The application of 200

MPa may not be high enough to allow full extraction of carotenoid. The increase in total carotenoid

179

observed in all other samples after high pressure treatment may be due to mechanical disruption

of the cell wall resulting in releasing free carotenoids into the juice. In fact, high pressure along

with nisin and a slight increase in temperature affects membrane integrity, creating pores in the

cell membrane through which carotenoid molecules may be released. Many studies show an

increase in carotenoid content after high pressure treatments. Hsu (2008) observed an increase up

to 62% in carotenoid content for tomato purée treated with a minimum pressure of 300 MPa;

Sánchez‐Moreno et al. (2006) observed an increase in carotenoid of tomato purée treated by HPP

at 400 MPa and 25°C for 15 min.

The short treatment duration (2 minutes), the moderate temperature, pressure, and nisin

levels used here may explain why the processing conditions did not affect the carotenoid content,

whereas other studies reported changes with statistical significance. For example, Patras et al.

(2009a) reported a significant increase in carotenoid content in carrot purée after HPP treatment at

400, 500, and 600 MPa for 15 minutes (58% of increase at 600 MPa).

3.5.2 Effect on total phenolic compounds

The results presented in Table 20 show an increase in the concentration of phenolic compounds in

all treated samples relative to the untreated carrot juice. Nevertheless, this increase was not

significant, which may be due to the mild conditions used in this study. As with carotenoids, this

may be explained by the membrane damages caused by the combination of HPP, nisin, and

temperature (see 3.5.1). Our observations follow the same trend described by Patras et al. (2009b),

who reported a 9.8% increase in the phenolic content of strawberry purée treated at 600 MPa.

Barba et al. (2013) reported similar results: a 13 to 27% increase in blueberry juice samples treated

180

by HPP at 200 MPa for 5 to 15 minutes, and a 24% increase when they were treated at 400 MPa

for 15 min.

Table 20. Effect of selected combination of treatments on carotenoids, phenolic compounds and ascorbic

acid

Samples

Carotenoids

(mg/L)

Phenolics

(mg/L)

Ascorbic Acid

(mg/100g juice)

a. Control 435.18±0.00a 26.13±0.91a 23.12±2.86a

b. 200 MPa, 50ppm nisin, 50°C 394.73±15.00a 28.28±0.87a 16.73±1.76a

c. 300 MPa, 25ppm nisin, 35°C 465.18±0.00a 27.26±0.34a 19.15±3.30a

d. 400 MPa, 0ppm nisin, 35°C 450.09±50.00a 28.64±1.19a 18.49±0.00a

e. 500 MPa, 0ppm nisin, 20°C 515.63±48.6a 27.77±1.16a 18.49±0.00a

The values are a mean ± standard deviation. The same letter (superscript) after standard deviation in the

column indicate no significant difference (p>0.05) between the samples/processing conditions.

3.5.3 Effect on ascorbic acid

The study of the retention of ascorbic acid in carrot juice after processing reveals a non-significant

decrease in this vitamin in each treated sample compared to the unprocessed control (Table 20).

Although the mechanism of ascorbic acid degradation is not completely elucidated, researchers

have observed two different paths in ascorbic acid degradation. One follows an aerobic path, and

the other a non-anaerobic path, described by Burdurlu et al. (2006).

In the aerobic mechanism, the ascorbic acid reacts with O2 at the interface between the air

and the juice. In addition, according to Henry’s Law:

181

𝑃𝑂2 = 𝐻 × 𝑥𝑂2 ,

where PO2 is the pressure due to O2 in the air in Pa, χO2 the mole-proportion of O2 in the solution,

and H the Henry’s constant of 769.2 L.atm/mol. It can be seen that an increase in pressure leads to

an increase in the proportion of dissolved O2 in the solution. In the case of HPP, the increase in

pressure may lead to an increase in the O2 solubility; according to Robertson and Samaniego

(1986), the rate of oxidative ascorbic acid degradation depends on O2 dissolution. Therefore, the

more O2 is in the solution; the more ascorbic acid is oxidized.

This study also reveals that high-pressure treatment of carrot juice at 50°C can lead to a

higher reduction of ascorbic acid compared to the treatment at 20 or 35°C. This may be due to the

fact ascorbic acid is a precursor of non-enzymatic degradation, and this reaction is accelerated at

high temperature. The treatment only lasted two minutes, which may not be long enough to allow

the reaction to be completed. These results are consistent with the ones obtained by Patras et al.

(2009b), who demonstrated that thermally processed strawberry purée contains less ascorbic acid

than a sample treated with HPP. The same authors observed that thermal processing (70°C, 2 min)

led to a decrease of 22.6% in ascorbic acid in strawberry purée, while a 600 MPa treatment led to

only a 6% reduction in ascorbic acid levels. In any case, in the present study, the decrease in

ascorbic acid at 50°C is not significant, potentially explained by the relatively small increase in

temperature and short duration of this experiment compared to the more intense conditions

described by Patras et al. (2009b). The results of the present work are in line with the findings of

Fernández-García et al. (2001) who observed a non-significant decrease in ascorbic acid in orange-

lemon-carrot juice treated by HPP at 500 MPa and 800 MPa for 5 minutes.

182

3.6 Effect of process on energy consumption

Statistical analysis of the energy consumed during each process reveals that rising the pressure at

constant temperature increases the energy required significantly (p<0.05). Results are presented in

Table 21.

Raising the pressure from 200 MPa to 500 MPa cost more energy than raising the

temperature of the high pressure vessel fluid from 20°C to 50°C. Raising the vessel’s pressure

implies compression work and compression heating of both the sample and the vessel fluid. Hence,

heating is responsible for 55% of the energy consumed during the process at 200 MPa, 50°C, but

only 20% for the process performed at 300 MPa, 35°C, and 13% for the one at 400 MPa, 35°C. It

is important to keep in mind that these data refers to the HPP equipment used in this study.

Table 21. Compression work and total energy consumption during processing

Processing conditions Wcompression (kJ/L) Total energy (kJ/L)

b: 200 MPa, 50°C 99.4 224.6a

c: 300 MPa, 35°C 249.4 312.0c

d: 400 MPa, 35°C 423.7 486.3b

e: 500 MPa, 20°C 541.7 541.7d


the column indicate no significant difference (p>0.05) between the samples/processing conditions.

3.7 Storage studies

A product shelf life can be defined as the time during which it can be consumed safely. The shelf

life of minimally processed juice is generally about 15-20 days. Therefore, the selected processing

183

conditions have to maintain a low level of microbial load during storage period as well as quality

attributes. Hence, microbial load, pH, TSS, turbidity, and color were analyzed for 28 days at 4°C

3.7.1 Microbiological aspects

The steady growth of total aerobic mesophilic bacteria was observed in all samples during storage

at 4°C for 4 weeks (Figure 4). The control sample was spoiled with total mesophilic count over 5

log when the juice was analyzed after one week. Sample c (300 MPa, 35°C, 25 ppm nisin) had the

lowest microbial count at week zero (just after processing) and has only less than 2 log CFU/mL

of mesophilic bacteria after 28 days of storage. The sample b (200 MPa, 50°C, and 50 ppm nisin)

was also microbiologically stable for 4 weeks. Samples d and e are those without nisin the bacterial

load was around 1 log just after processing whereas on day 28, the microbial load was around 3-

log. The comparison between carrot juice with and without nisin shows that both types of juice

had almost equivalent load of aerobic bacteria just after processing, but during storage, juice with

nisin inhibited growth, making the juice more stable than the juice without it.

Sample c which contains 2 log of bacteria at day 28, was considered as microbiologically

stable for four weeks, a good shelf life for this type of low-acid minimally processed product. The

incorporation of 25 ppm nisin and processing at 35°C made the juice microbiologically safe for 4

weeks at 4°C without using significant high pressures (≥500 MPa) or high temperature treatment

(>70°C), or using chemical preservatives.

184

Figure 28. Growth of aerobic mesophilic bacteria in carrot juice during storage at 4°C when exposed to

different treatments: b: 200 MPa, 50 ppm, 50°C, c: 300 MPa, 25 ppm, 35°C, d: 400 MPa, 0 ppm, 35°C, e:

500 MPa, 0 ppm, 20°C.

In the study on the combined effect of HPP and nisin on cucumber juice conducted by Zhao

et al. (2013), the authors found that in juice treated by 500 MPa for 2 min with nisin (100 IU/mL),

the total aerobic bacteria count grew more slowly than in juice treated without nisin during storage

at 4°C for 50 days. The total aerobic bacteria in juice treated with 500 MPa, 2 min and 100 IU/mL

nisin was just 1.5 log cycles at the end of the storage period. The authors claimed that the cucumber

juice treated with nisin was microbiologically safe for 50 days at 4°C. In the present study, it can

be claimed that application of 300 MPa of pressure with 25 ppm nisin, and processing at 35°C for

2 min was able to make carrot juice microbiologically stable for 4 weeks at 4°C. Level of pressure

in the present study was much lower than Zhao’s work (2013), hence, the shelf life is shorter.

Application of higher pressure keeping nisin concentration and processing temperature constant

would definitely render a longer shelf life for carrot juice than the one reported here.

0

0.5

1

1.5

2

2.5

3

3.5

4

0 1 2 3 4

Log

(CFU

/mL)

Weeks

b

c

d

e

185

3.7.2 Change in pH, total soluble solids and turbidity during storage

There was a steady and significant (p<0.05) reduction of pH, soluble solids, and turbidity in fresh

carrot juice (control sample) during storage. Initially, the pH value of fresh carrot juice was 6.60.

After storage at 4°C for 4 weeks, its pH had dropped to 4.82. In the carrot juice treated with 50

ppm nisin and 200 MPa at 50°C (sample a), there was also a significant reduction of pH in week

4, but the reduction of pH was not significant until week 3. For sample c (25 ppm nisin, 300 MPa

pressure, 35°C), there was a slight reduction of the pH across the whole storage period. The same

trend was observed in sample d which was treated by 400 MPa, 35°C (without nisin) as well as in

the sample ‘e’ treated by 500 MPa, 20°C. The reduction of pH in control sample during storage

might be due to the production of acids by microorganisms (Palma-Harris et al., 2002). In samples

with nisin, the change in pH during storage is less significant (Table 22), likely due to the presence

of fewer aerobic mesophilic bacteria generating acid by-products.

Slight decreases in total soluble solids were observed in the control sample during storage,

but in all of the processed samples, there was non-significant (p>0.05) reduction. This observation

may suggest that the sensory quality may stay unchanged after 4 weeks of storage at 4°C.

Regarding turbidity, there was a gradual reduction in the control sample which might be associated

with microbial growth. In all the processed samples, the reduction is not steady, and the change is

non-significant (p>0.05).

186

Table 22. Change in pH, Total soluble solids and Turbidity of carrot juice during storage at 4°C.

Weeks pH TSS (°Bx) Turbidity (Abs. at 660 nm)

a: Control

Sample

(Untreated

fresh carrot

juice)

0 6.60±0.00a 9.35±0.15a 2.32±0.03a

1 5.62±0.03ab 8.40±0.00ab 2.14±0.02a

2 5.55±0.41ab 7.85±0.05b 1.94±0.31a

3 5.89±0.03a 7.85±0.05b 1.04±0.13b

4 4.82±0.02b 7.70±0.40b 0.66±0.04b

b: 200 MPa,

50 ppm

nisin, 50°C

0 6.40±0.01a 8.30±0.00a 2.09±0.01a

1 6.08±0.10a 8.60±0.10a 2.06±0.00ab

2 6.35±0.02a 8.05±0.25a 1.75±0.22ab

3 6.38±0.01a 7.85±0.45a 1.67±0.02b

4 5.53±0.12b 8.30±0.00a 2.14±0.01a

c: 300 MPa,

25 ppm,

35°C

0 6.43±0.00a 8.10±0.00a 2.02±0.02a

1 6.22±0.01b 8.25±0.05a 1.53±0.27ab

2 6.06±0.01c 8.15±0.05a 1.52±0.00ab

3 6.04±0.01c 8.20±0.00a 1.34±0.06b

4 6.03±0.01d 8.15±0.05a 1.46±0.13ab

0 6.44±0.00ab 8.50±0.10a 2.01±0.05ab

187

d: 400 MPa,

0 ppm

nisin, 35°C

1 6.26±0.00ab 8.60±0.10a 1.79±0.06bc

2 6.04±0.03b 8.45±0.05a 1.77±0.09bc

3 6.64±0.01a 8.80±0.10a 1.69±0.04c

4 6.09±0.22ab 8.65±0.15a 2.14±0.02a

e: 500 MPa,

0 ppm,

20°C

0 6.41±0.00ab 9.05±0.05a 2.04±0.05a

1 6.16±0.01cd 9.15±0.15a 1.93±0.17a

2 6.02±0.02d 8.90±0.10a 1.76±0.10a

3 6.51±0.05a 9.00±0.10a 1.74±0.05a

4 6.27±0.01bc 8.90±0.00a 2.10±0.00a



3.7.3 Change in color characteristics

There was no significant difference (p>0.05) in color of carrot juice between control and treated

sample immediately after processing. A gradual reduction in lightness (L*) value was observed in

all the samples including the control. López-Malo et al. (1998) found no significant changes in the

L* values of high-pressure processed (345 MPa and 689 MPa) avocado purée during storage at

5°C.

In terms of color difference (ΔE*), no significant changes were observed in any high

pressure processed samples until three weeks of storage at 4°C, but in control samples, the ΔE

value changed very rapidly from the second week. Non-significant changes in the ΔE* value in

188

processed samples indicates that color of the carrot juice was well-preserved by HPP. The Chroma

value (C*) did not change significantly in sample c, d, and e for three weeks. In the case of sample

b, it was stable for two weeks, but in the control sample Chroma value was stable for only one

week. There was a gradual reduction in hue angle (h*) in all samples during the storage period.

There was no difference in hue angle between different processing conditions.

Table 23. Change in color characteristics of carrot juice during storage at 4°C.

Weeks Lightness (L*) ΔE* Chroma (C*) Hue Angle (h*)

a: Control

Sample

(Untreated

fresh carrot

juice)

0 12.09±0.41a 38.63±0.95a 0.56±0.01a

1 9.88±0.68a 4.47±2.08a 37.33±0.86a 0.47±0.04ab

2 5.38±0.44b 11.91±0.01b 30.90±0.96b 0.39±0.01bc

3 6.88±0.36b 14.86±0.37b 26.54±1.72c 0.35±0.01c

4 5.67±0.01b 15.71±1.26b 25.84±0.32c 0.36±0.02c

b: 200 MPa,

50 ppm nisin,

50°C

0 10.86±0.62a 36.78±1.40a 0.53±0.02a

1 7.59±0.13b 6.83±1.71a 32.53±0.56ab 0.41±0.00a

2 6.06±0.40bc 6.81±2.81a 33.97±1.21a 0.43±0.07a

3 3.64±0.58c 18.9±3.92a 26.00±3.73b 0.63±0.44a

4 5.96±0.85bc 13.1±2.29a 25.25±0.50b 0.42±0.07a

c: 300 MPa,

25 ppm, 35°C

0 11.03±0.15a 36.68±0.34a 0.54±0.00a

1 7.69±0.84ab 7.22±0.88a 35.50±3.79a 0.38±0.02bc

189

2 7.24±0.13ab 7.89±0.21a 31.74±1.55ab 0.40±0.01bc

3 5.50±1.78b 8.36±0.73a 31.62±0.46ab 0.42±0.02b

4 5.18±0.09b 13.6±0.68b 25.95±0.55b 0.35±0.00c

d: 400 MPa,

0 ppm nisin,

35°C

0 10.66±0.67a 35.79±1.72a 0.53±0.02a

1 8.61±0.22ab 6.53±3.36a 31.91±1.57a 0.39±0.08a

2 7.41±0.31ab 6.90±2.20a 31.20±0.74a 0.41±0.01a

3 6.39±0.93b 9.04±3.72a 30.19±3.07a 0.37±0.04a

4 6.28±0.87b 10.06±0.67a 28.00±2.66a 0.39±0.04a

e: 500 MPa, 0

ppm, 20°C

0 8.98±1.22a 33.30±2.52a 0.48±0.06a

1 7.37±0.03ab 3.66±2.22a 30.90±0.65ab 0.41±0.00a

2 6.42±0.54ab 5.57±1.33a 29.43±1.36ab 0.38±0.02a

3 6.26±0.24ab 5.70±3.20a 29.67±0.88ab 0.38±0.01a

4 4.37±0.99b 10.12±1.24a 26.92±1.28b 0.28±0.10a



4. Conclusions

The use of HPP in conjunction with nisin and moderate thermal treatments is an effective way of

reducing microbial load in carrot juice, and potentially in other low-acid juices. The addition of

nisin and processing at mild temperatures (35 or 50°C) enhanced the destruction of both gram-

190

positive and gram-negative bacteria. The combined effect of nisin, mild temperatures and HPP

was better to inactivate bacteria than using one hurdle at a time due to their complementarity

actions on membrane cell. These results demonstrated that the proposed combination would

effectively reduce the microbial loads in low-acid juices using moderate pressure and temperature,

which could significantly reduce processing cost and increase throughput. For instance, treatment

performed at 300 MPa, 25 ppm of nisin at 35°C could be an effective combination of hurdles. This

leads to at least 5 log reductions of both E. coli and Listeria innocua, and was stable for 4 weeks

in terms of aerobic mesophilic bacterial growth, color, TSS, turbidity, and did not have any

significant effect on the degradation of ascorbic acid, phenolic compounds, and carotenoid content.

High pressure treatment in combination with nisin and mild temperatures seems to be effective

and offer cost savings by increasing energy efficiency and throughput. These types of combined

treatments can greatly reduce energy requirements with no loss of efficacy compared to

conventional thermal processes or HPP when used alone.

191

References

Barba, F. J., Esteve, M. J., & Frigola, A. (2013). Physicochemical and nutritional characteristics

of blueberry juice after high pressure processing. Food Research International, 50(2), 545-

549.

Bermúdez-Aguirre, D., Corradini, M. G., Candoğan, K., & Barbosa-Cánovas, G. V. (2016). High

Pressure Processing in Combination with High Temperature and Other Preservation

Factors. In High Pressure Processing of Food (pp. 193-215). Springer New York.

Bos, M. P., Robert, V., & Tommassen, J. (2007). Biogenesis of the gram-negative bacterial outer

membrane. Annual Review of Microbiology, 61, 191-214.

Boziaris, I. S., & Adams, M. R. (1999). Effect of chelators and nisin produced in situ on inhibition

and inactivation of Gram negatives. International Journal of Food Microbiology, 53(2),

105-113.

Brewer, R., Adams, M. R., & Park, S. F. (2002). Enhanced inactivation of Listeria monocytogenes

by nisin in the presence of ethanol. Letters in Applied microbiology, 34(1), 18-21.

Burdurlu, H. S., Koca, N., & Karadeniz, F. (2006). Degradation of vitamin C in citrus juice

concentrates during storage. Journal of Food Engineering, 74(2), 211-216.

Ciancaglinia, P., Santos, H. L., Daghastanli, K.R.P., Thedei , Gerald Jr. (2001). Using a classical

method of vitamin C quantification as a tool for discussion of its role in the body.

Biochemistry and Molecular Biology Education. 29(3), 110-114

192

de Arauz, L. J., Jozala, A. F., Mazzola, P. G., & Penna, T. C. V. (2009). Nisin biotechnological

production and application: a review. Trends in Food Science & Technology, 20(3), 146-

154.

Dede, S., Alpas, H., & Bayındırlı, A. (2007). High hydrostatic pressure treatment and storage of

carrot and tomato juices: Antioxidant activity and microbial safety. Journal of the Science

of Food and Agriculture, 87(5), 773-782.

Derradji-Benmeziane, F., Djamai, R., & Cadot, Y. (2014). Antioxidant capacity, total phenolic,

carotenoid, and vitamin C contents of five table grape varieties from Algeria and their

correlations. OENO One, 48(2), 153-162.

Fernández García, A., Butz, P., Bognàr, A., & Tauscher, B. (2001). Antioxidative capacity,

nutrient content and sensory quality of orange juice and an orange-lemon-carrot juice

product after high pressure treatment and storage in different packaging. European Food

Research and Technology, 213(4), 290-296.

Hauben, K. J., Wuytack, E. Y., Soontjens, C. C., & Michiels, C. W. (1996). High-pressure transient

sensitization of Escherichia coli to lysozyme and nisin by disruption of outer-membrane

permeability. Journal of Food Protection®, 59(4), 350-355.

Helander, I. M., & Mattila-Sandholm, T. (2000). Permeability barrier of the Gram-negative

bacterial outer membrane with special reference to nisin. International Journal of Food


Hsu, K. C. (2008). Evaluation of processing qualities of tomato juice induced by thermal and

pressure processing. LWT-Food Science and Technology, 41(3), 450-459.

193

Jack, R. W., Tagg, J. R., & Ray, B. (1995). Bacteriocins of gram-positive bacteria. Microbiological

Reviews, 59(2), 171-200.

Kalchayanand, N., Dunne, P., Sikes, A., & Ray, B. (2004). Viability loss and morphology change

of foodborne pathogens following exposure to hydrostatic pressures in the presence and

absence of bacteriocins. International Journal of Food Microbiology, 91(1), 91-98.

Lee, D. U., Heinz, V., & Knorr, D. (2003). Effects of combination treatments of nisin and high-

intensity ultrasound with high pressure on the microbial inactivation in liquid whole

egg. Innovative Food Science and Emerging Technologies, 4(4), 387-393.




López-Malo, A., Palou, E., Barbosa-Cánovas, G. V., Welti-Chanes, J., & Swanson, B. G. (1998).

Polyphenoloxidase activity and color changes during storage of high hydrostatic pressure

treated avocado puree. Food Research International, 31(8), 549–556.

Lubelski, J., Rink, R., Khusainov, R., Moll, G. N., & Kuipers, O. P. (2008). Biosynthesis,

immunity, regulation, mode of action and engineering of the model lantibiotic

nisin. Cellular and Molecular Life Sciences, 65(3), 455-476.

McNamee, C., Noci, F., Cronin, D. A., Lyng, J. G., Morgan, D. J., & Scannell, A. G. M. (2010).

PEF based hurdle strategy to control Pichia fermentans, Listeria innocua and Escherichia

coli k12 in orange juice. International Journal of Food Microbiology, 138(1), 13-18.

194

Murchie, L. W., Cruz-Romero, M., Kerry, J. P., Linton, M., Patterson, M. F., Smiddy, M., & Kelly,

A. L. (2005). High pressure processing of shellfish: a review of microbiological and other

quality aspects. Innovative Food Science and Emerging Technologies, 6(3), 257-270.

Oey, I., Van der Plancken, I., Van Loey, A., & Hendrickx, M. (2008). Does high pressure

processing influence nutritional aspects of plant based food systems?. Trends in Food


Palma-Harris, C., McFeeters, R. F., & Fleming, H. P. (2002). Fresh cucumber flavor in refrigerated

pickles: Comparison of sensory and instrumental analysis. Journal of Agricultural and

Food Chemistry, 50, 4875–4877.

Patras, A., Brunton, N. P., Da Pieve, S., & Butler, F. (2009b). Impact of high pressure processing




Patras, A., Brunton, N., Da Pieve, S., Butler, F., & Downey, G. (2009a). Effect of thermal and high

pressure processing on antioxidant activity and instrumental colour of tomato and carrot

purées. Innovative Food Science and Emerging Technologies, 10(1), 16-22.

Plaza, L., Sánchez-Moreno, C., Elez-Martínez, P., de Ancos, B., Martín-Belloso, O., & Cano, M.

P. (2006). Effect of refrigerated storage on vitamin C and antioxidant activity of orange

juice processed by high-pressure or pulsed electric fields with regard to low

pasteurization. European Food Research and Technology, 223(4), 487-493.

195

Robertson, G. L., & Samaniego, C. M. L. (1986). Effect of initial dissolved oxygen levels on the

degradation of ascorbic acid and the browning of lemon juice during storage. Journal of

Food Science, 51(1), 184-187.

Sánchez‐Moreno, C., Plaza, L., De Ancos, B., & Cano, M. P. (2006). Impact of high‐pressure and

traditional thermal processing of tomato purée on carotenoids, vitamin C and antioxidant

activity. Journal of the Science of Food and Agriculture, 86(2), 171-179.

Shiu, C., Zhang, Z., & Thomas, C. R. (2001). A comparison of the mechanical properties of

different bacterial species. In Applied Microbiology (pp. 155-162). Springer Netherlands.

Silva, F. V. (2016). Modeling the inactivation of psychrotrophic Bacillus cereus spores in beef

slurry by 600MPa HPP combined with 38–70° C: Comparing with thermal processing and

estimating the energy requirements. Food and Bioproducts Processing, 99, 179-187.

Smith, K., Mittal, G. S., & Griffiths, M. W. (2002). Pasteurization of milk using pulsed electrical

field and antimicrobials. Journal of Food Science, 67(6), 2304-2308.

Sokołowska, B., Skąpska, S., Fonberg-Broczek, M., Niezgoda, J., Chotkiewicz, M., Dekowska,

A., & Rzoska, S. (2012). The combined effect of high pressure and nisin or lysozyme on

the inactivation of Alicyclobacillus acidoterrestris spores in apple juice. High Pressure

Research, 32(1), 119-127.

ter Steeg, P. F., Hellemons, J. C., & Kok, A. E. (1999). Synergistic actions of nisin, sublethal

ultrahigh pressure, and reduced temperature on bacteria and yeast. Applied and

Environmental Microbiology, 65(9), 4148-4154.

196

Tong, Z., Ni, L., & Ling, J. (2014). Antibacterial peptide nisin: A potential role in the inhibition

of oral pathogenic bacteria. Peptides, 60, 32-40.

Wuytack, E. Y., Diels, A. M., & Michiels, C. W. (2002). Bacterial inactivation by high-pressure

homogenisation and high hydrostatic pressure. International Journal of Food


Zhang, Y., Liu, X., Wang, Y., Zhao, F., Sun, Z., & Liao, X. (2016). Quality comparison of carrot

juices processed by high-pressure processing and high-temperature short-time

processing. Innovative Food Science and Emerging Technologies, 33, 135-144.

Zhao, L., Wang, S., Liu, F., Dong, P., Huang, W., Xiong, L., & Liao, X. (2013). Comparing the

effects of high hydrostatic pressure and thermal pasteurization combined with nisin on the

quality of cucumber juice drinks. Innovative Food Science and Emerging

Technologies, 17, 27-36.

197

CHAPTER SIX

INACTIVATION OF Listeria innocua IN CARROT-ORANGE JUICE BLENDS BY

HIGH HYDROSTATIC PRESSURE

Abstract

High Pressure Processing (HPP) is a nonthermal technology that has shown promising results as

an alternative to thermal pasteurization of fruit and vegetable juices. The objective of this study

was to develop a processing method to effectively pasteurize carrot-orange juice blends of different

pH using mild HPP treatment and short processing time. The effect of three different pH levels on

the inactivation of L. innocua (ATCC 8456TM) was studied at three levels of pressure and five

different treatment times at room temperature (20°C).

For the juice blend with pH 4, 300 MPa of pressure and 2 min of treatment time was

required to achieve more than 6 log reduction of L. innocua. For the same level of inactivation,

400 MPa, 1 min was required for the 1:1 blend (pH 5), and for pH 6, 400 MPa, 3 min was required.

In case of treatments with 400 MPa, there was complete inactivation (7 log reduction) in the blends

with pH 4 and 5 in one minute; but in the case of pH 6, three min was required to achieve the same

level of inactivation.

Storage studies show that the selected HPP conditions were able to maintain the aerobic

mesophilic bacteria count below 2 log for 28 days. There were no significant changes in PH, color,

and TSS during storage for the juice with pH 4 and 5. For juice with pH 6, slight change on those

characteristics were observed during storage.

198

These results show that HPP can be used to safely process selected carrot-orange juice

blends without sacrificing quality attributes. Addition of orange juice to carrot juice is a natural

way to add hurdles that can play a significant role in the reduction of pressure and treatment time

which finally reduces the energy input and processing cost.

1. Introduction

Fresh fruit and vegetable juices are excellent sources of essential bioactive compounds. In the

current market, the mixture of fruit and vegetable juices is very popular as it can balance the

nutrients and enhance the taste. Carrot-orange juice blend is rich in beta carotene (from carrot) and

ascorbic acid (from orange) and therefore, it is a very popular beverage in the United States

because of its high nutritive value. The addition of orange juice in carrot juice not only balance the

nutrient but also increases the acidity making the product suitable for processing. Mixing orange

juice with carrot juice is therefore a good way to lower the pH and still comply with clean label

requirements. During thermal processing and storage of such juice, significant losses in valuable

nutrients as well as changes in microbial quality and physicochemical characteristics can take place

(Arena et al., 2001; Polydera et al., 2003). Thermal processing is typically conducted at the

temperature higher than 60°C for a few seconds to minutes based on the pH of juice. High

temperature and longer holding time during processing have shown to have detrimental effects on

the quality of juice and blends (Chen et al., 2013). Food scientists and engineers are now

developing novel methods to process nutritious beverages to preserve their valuable nutrients and

making the product microbiologically safe. To better optimize each process, researchers are also

working to improve process efficiency and reduce operation cost.

199

High pressure processing is one of the novel methods that is currently expanding in the

field of beverage processing. During processing by HP there is generation of small amount of heat

due to compression, which positively impacts the inactivation of microorganisms. Since

temperature is not the governing factors in HPP, there is greater chances of retention of nutrients

during processing (Oey et al., 2008). Additionally, HPP does not affect covalent bonds, hence,

nutrients are not much affected. Moreover, the process is independent of the shape and the size of

the products, but its effect on microorganisms depends on the pH and water activity of the product.

Treating product with high pH (>6.5) using only HPP, requires a very high pressure (>600 MPa)

and a long holding time (>5 min). These two requirements make the process less efficient and less

cost effective. Hence, some modifications of the product or the process are necessary to enhance

the HPP treatment. Since pressure and pH can synergistically inactivate microorganisms present

in the product, lowering the pH could decrease the pressure and/or the treatment time.

The main objective of this study was to investigate the effect of different pH of juice on

the inactivation of microorganisms by HPP. The approach was to combine fruit and vegetable

juices to have a blend with different pH and to process it by HPP using mild-pressures, and short

holding times with the aim of reducing energy input. Another objective of this study was to explore

the effect of HPP on the physicochemical characteristics of blended juice immediately after

processing and during storage at 4°C for 28 days. The overall aim of this study was to develop a

processing methodology based on the pH of juice blends, level of pressure, and treatment time to

predict the microbial inactivation, energy consumption and shelf life of the products.

200


This section discusses the method of preparation of juice, inoculation of Listeria culture, and the

protocol used for the processing. Additionally, the methods used for microbiological and

physicochemical analysis are highlighted.

2.1 Sample Preparation

In the first part of the study, three juices with pH 4, 5, and 6 were prepared using pre-pasteurized

carrot juice (Boalthouse Farms, Bakersfield, CA) and orange juice (Simply Orange Juice

Company, Apopka, FL). Three different formulations were created using Pearson’s square method

in which carrot juice with pH 6.4 was mixed with orange juice with pH 3.5 to get blends with

various pH. The proportion of carrot and orange in the blends were 1:6, 1:1, and 6:1 for pH 4, 5,

and 6 respectively. Since there was no competitive microflora present in both pasteurized juices,

the samples were used to select the level of pressure and time for each pH that can result in at least

five log reduction of L. innocua.

After the completion of microbial inactivation studies, fresh carrot juice and fresh orange

juice were prepared in the lab using a domestic juice extractor (Hamilton Beach model 67608Z,

Hamilton Beach Brands, Inc., Glen Allen, VA). The juices were mixed using the same method to

get the blends with pH 4, 5, and 6. Fresh juice was used in this phase to study the effect of high

pressure on the selected quality characteristics immediately after processing and during storage.

201

2.2 Culture Preparation and Inoculation

A freeze-dried culture of L. innocua cells (ATCC 51742TM) was grown using 100 ml sterilized

tryptic soy broth (Becton, Dickinson and Co., Sparks, MD) with 0.6% yeast extract (Becton,

Dickinson and Co., Sparks, MD). Cells of Listeria were kept in a water bath shaker at 37°C and

218 rpm until the early stationary phase was reached. The growth curve was plotted by measuring

the absorbance at 540 nm every 30 minutes using a spectrophotometer (Spectronic 20 Genesys,

Spectronic Instruments Inc., Rochester, NY). Cells were harvested at the early stationary phase

and vials with 2 ml of 20% glycerol and 2 ml of culture were stored at -21 °C until use. Frozen

culture was thawed during the experiment and activated using 100 ml sterilized tryptic soy broth

plus 0.6% yeast extract. Juice samples were inoculated with L. innocua to get the final

concentration of 7 log CFU/ml.

2.3 High Pressure Processing

The juice samples (20 mL) with culture were packaged into Nylon/PE pouches (3 Mil, Ultra

Source, Kansas City, MO) and sealed using the impulse sealer (Impulse Heat Sealer 3T06,

Midwest Pacific, Taiwan). Pouches were immediately pressurized using an Engineering Pressure

System Inc’s (EPSI) HPP unit. The desired pressure level was achieved using an electrohydraulic

intensifier pump (Hochdruck-Systeme GmbH, AP 10-0670-1116, Sigless, Austria) that

pressurized the vessel to operating pressure in a few seconds (5-10 seconds). Water with 5% Mobil

Hydrasol 78 was used as the pressure transmitting medium. Processing conditions were 200, 300,

and 400 MPa with a holding time of 1 to 5 min and the temperature before processing was 20°C.

After treatment, samples were placed in ice and further analysis was conducted immediately.

202

2.4 Analysis of Survivors

Microbiological analysis of samples was conducted immediately after processing. Juice samples

were serially diluted in sterile peptone water (0.1% Bacto™ Peptone, Difco Laboratories) and

pour-plating was conducted with Tryptic Soy Agar with 0.6% yeast extract (TSBYE). Plates were

incubated for 48 hours at 37°C and colony forming units (CFU)/ml were counted manually.

2.5 Determination of Color and Soluble Solid Contents

Color parameters of the blended juice samples were measured before and after processing. A

sample of juice blend (10 mL) was poured into the transparent cuvette and color parameters (L*,

a*, and b*) were determined using a Minolta CM-2002 colorimeter (Minolta Camera Co., Osaka,

Japan). Soluble solids content was assessed using a hand refractometer (Atago No. 3840, Atago,

Tokyo, Japan).

2.6 Ascorbic Acid

The ascorbic acid content of the juice blends was determined using the methods described by

Ciancaglini et al. (2001). This procedure uses iodine solution (0.005 mol/L) which reacts with

ascorbic acid to produce dehydroascorbic acid and iodide ions as follows:

𝑎𝑠𝑐𝑜𝑟𝑏𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝐼2 ⇾ 2𝐼− + 𝑑𝑒ℎ𝑦𝑑𝑟𝑜𝑎𝑠𝑐𝑜𝑟𝑏𝑖𝑐 𝑎𝑐𝑖𝑑

In brief, 20 mL of juice blends was added to 150 mL of water and 1 mL of starch solution (0.5%,

w/v). This solution was kept under agitation and iodine was added until the end point (the

formation of dark green color). The volume of iodine required to reach the end point was used to

203

determine the ascorbic acid concentration and was expressed in mg ascorbic acid equivalent per

100 g of juice.

2.7 Total Soluble Phenolic

Phenolic content in the juice before and after treatment was determined using the protocol

described by Derradji-Benmeziane et al., (2014). The carrot-orange juice blend (50 μL) was poured

into a volumetric flask; afterwards, 2.5 mL of Folin-Ciocalteu reagent (diluted 1/10) was added.

After leaving for 10 min at room temperature, 2 mL of Na2CO3 (7.5%) was added and the sample

was kept for 30 min in the dark. Absorbance was determined at 760 nm. The standard curve was

prepared using Gallic acid and the results were expressed in mg equivalent Gallic acid per L.

2.8 Total Carotenoid

Carotenoid content of the juice was determined using the method described by Liao et al., (2007)

with some modification. In brief, 25 mL of juice was poured into a separating funnel. Then 80 mL

of a hexane-acetone mix (1:1, v/v) were added directly in the separation funnel. The organic phase

was collected and anhydrous sodium sulfate was added to absorb the moisture. The absorbance

was measured at 450 nm at room temperature. A standard curve was prepared using β-carotene (2-

10 μg/mL) dissolved in hexane solution.

2.9 Quantification of Energy requirements

The total energy (W) in the system can be defined as the sum of energy required to increase the

pressure and the thermal energy which increases the temperature of the vessel (Silva et al., 2016),

𝑊 =1

2𝑉. 𝛽(𝑃𝑓𝑖𝑛𝑎𝑙

2 − 𝑃𝑖𝑛𝑖𝑡𝑖𝑎𝑙2) − 𝑃𝑓𝑖𝑛𝑎𝑙. 𝑉. 𝛼(𝑇𝑓𝑖𝑛𝑎𝑙 − 𝑇𝑖𝑛𝑖𝑡𝑖𝑎𝑙), (1)

204

𝜌. 𝐶𝑝.𝛿𝑇

𝛿𝑡= 𝑇. 𝛼𝑝.

𝛿𝑃

𝛿𝑡, (2)

where 𝛿𝑇

𝛿𝑡 and

𝛿𝑃

𝛿𝑡 are the variation of temperature and pressure over time respectively, V is the

volume of the cylinder in m3, P and T the pressure and the temperature in the vessel respectively,

β the compressibility (4.6E-10 Pa-1), α the volume expansivity (2.14E-4 K-1 or C-1) of the water

since the pressure transmitting fluid contains 95% of water.

The increases in pressure and temperature in the vessel are graphically shown in Figure 29.

The total energy used during the process corresponds to the energy involved during the come-up

time and can be therefore calculated by adding (1) and (2) after having collected the temperature

profile during each treatment. Results on energy consumption were expressed in kJ/L.

Figure 29. Temperature and pressure profile during processing at 400 MPa for 1 min used to determine

δT/δt and δP/δt required for the energy calculation

0

50

100

150

200

250

300

350

400

450

0

5

10

15

20

25

30

35

0 20 40 60 80 100 120 140

Pre

ssu

re (

MP

a)

Tem

per

atu

re (

°C)

Time (s)

Temperature (°C) Pressure (MPa)

205

2.10 Storage study

The shelf life study was performed only on selected mild conditions leading to at least 5 log

reduction of L. innocua. The main aim was to assess changes on physical characteristics and

mesophilic aerobic bacteria load in each sample during storage at 4°C for 28 days. After processing

juice blends with selected pressure and treatment time, they were stored for further analysis. Every

week juices were analyzed for pH, color, TSS, and mesophiles. Control samples (Unprocessed

juice) were also stored at 4°C and were analyzed every other day for 10 days. All the samples

were stored in the same pouches that were used to process, pouches had a water vapor transmission

rate of 6-7.5 g/m2.24 hr. and oxygen transmission rate of 50-70 cc/m2.24 hr.

2.11 Data Analysis

Data was processed using Microsoft excel 2016. Analysis of variance (ANOVA) between means

was carried out using SAS V. 9.2 (SAS Institute, Cary, NC, USA, 1999) at 5% level of

significance. Multivariate regression analysis was done using Minitab 16 (Minitab Inc. State

College, PA).


3.1 Inactivation of L. innocua

There was a significant impact of the pressure level, the pH of the product, and the processing time

on the inactivation of L. innocua. For the pH 4 blend, 300 MPa of pressure and 2 min of treatment

time were required to achieve more than 6 log reduction of L. innocua. For the same level of

inactivation, for pH 5, 400 MPa, 1 min was required and for pH 6, 400 MPa, 3 min was required.

206

Even after 5 minutes, the application of 200 MPa for the juices with pH 5 and 6 led to less than 2

log reduction and for the juice with pH 4 there was about 3 log reduction. In the case of treatment

at 400 MPa, Listeria count was below the detectable limit (7 log reduction) in the juice with pH 4

and 5 in one minute; but in the case of pH 6, three minutes were required to achieve the same level

of inactivation.

Figure 30. Inactivation of L. innocua in carrot-orange juice blends at 200 MPa, 300 MPa and 400 MPa

over 5 minutes

-4

-3

-2

-1

0

0 1 2 3 4 5

Log

(Nt/

N0)

Time (min)

200 MPa

pH4 pH 5 pH 6

-8

-6

-4

-2

0

0 1 2 3 4 5

Log

(Nt/

N0)

Time (min)

300 MPa

pH4 pH 5 pH 6

-8

-6

-4

-2

0

0 0.5 1 1.5 2 2.5 3

Log

(Nt/

N0)

Time (min)

400 MPa

pH4 pH 5 pH 6

207

According to the pressure-time combination applied to each blend to achieve at least 5 log

reduction, there was a synergistic interaction (p<0.05) between pressure and pH, pressure and time,

and pH and time. The results show that decreasing the pH reduces the pressure and/or the time of

treatment. Therefore, it minimizes energy consumption, as the energy required directly depends

on the level of pressure used.

The application of high pressure significantly inactivates L. innocua, as shown by this study

and a study performed by Evrendilek et al. (2011), who found that HPP performed at 600 MPa for

5 min leads to a reduction in L. innocua from 6 log CFU/mL to 1 log CFU/mL on ayran yogurt

(pH 4.2). HPP inactivates microorganisms by affecting protein structure, by changing internal pH

equilibrium (Balasubramaniam et al., 2015) and by creating pores in their membranes. Even

though HPP doesn’t affect covalent bonds, it affects weak bonds which can change the protein

conformation and may irreversibly damage the protein. Also, this change in conformation may

lead to decrease the rate of physiological reactions such as metabolism or replication reactions

(Sahu, 2014). According to Le Chatelier’s principle, the rise in pressure lead to a decrease in

volume. Bacterial cells contain vacuoles; when they are subjected to high pressure, lengthening of

the cell take place causing membrane’s deformation and formation of pores. Therefore, the longer

the treatment time is, the more bacterial cells are damaged and therefore the better the microbial

inactivation is.

A study performed by Conner et al. (1986) on cabbage juice shows that even if L.

monocytogenes can grow in a limited proportion and for a short time at low pH (pH<4.8), they

can’t survive for long period in such pH conditions and they can’t adapt to low pH. Therefore, low

208

pH could act as a hurdle in high pressure processing. According to the present study, combining

HPP and low pH results in a higher inactivation of L. innocua; even if the pH was reduced by 1

unit. High level of inactivation at low pH might be due to the formation of pores in the cell

membrane allowing the diffusion of protons and other acids from the environment to the

cytoplasm, which decreases the internal pH of the cell significantly. If the treatment time is long

enough, bacteria can’t recover from all these damages which lead to their death.

3.2 Multivariate Analysis

The effect of various factors on L. innocua inactivation was analyzed using multiple regression

method. The major factors involve are pressure, treatment time, and product pH. There was

significant effect of each factor on microbial inactivation. The main effect of pressure was highest

among three factors, the second was time and third pH (Table 24). There was interaction between

pressure-time, pH-pressure, and pH-time on the inactivation of this microorganism. The

interaction between pressure and pH had highest weight followed by pressure and time interaction.

Three-way interaction was also observed between all three factors.

Table 24. Main effect and interaction effect of each factor on L. innocua inactivation

Factors Total

Sensitivity

Main

Effect

pH

Interaction

Pressure

Interaction

Time

Interaction

pH 0.075 0.034 . 0.038 0.003

Pressure 0.882 0.814 0.038 . 0.031

Time 0.105 0.071 0.003 0.031 .

Multivariate regression relation was developed between all three factors to predict the inactivation

of L. innocua in the juice blend (Eq. 3):

209

𝐿𝑜𝑔 (𝑁0

𝑁𝑡) = −0.6 + 0.04 𝑝𝐻 + 0.03 𝑃 + 2.14 𝑡 − 0.003 𝑝𝐻. 𝑃 − 0.63 𝑝𝐻. 𝑡 − 0.006 𝑃. 𝑡 + 0.002 𝑝𝐻. 𝑃. 𝑡, (3)

where P is pressure in MPa and t is time in min.

Using this relation, processing parameters for HPP can also be approximately predicted

based on target level of Listeria inactivation in a juice with certain pH.

3.3 Energy consumption during processing

Total energy consumed during each process is presented in Table 25. The results include the

contribution in energy consumption during come up time. It was observed that the thermal energy

generated by the adiabatic compression during the come-up time contributes about 10% or more

on the total energy consumed by the system. Hence, some of the energy is converted into heat as

a result of pressure increase. As expected, the energy consumed in the process run at 300 MPa is

about 1.8 times lower than the others run at 400 MPa. Moreover, the energy consumed during

experiments set at 400 MPa for 1 min is fairly similar to the one consumed at 400 MPa for 3 min.

Table 25. Energy consumption in HPP at different pressure and processing time

Juice blends Pressure (MPa)

Holding

time (min)

Energy due to

Compression (kJ/L)

Total Energy

(kJ/L)

Carrot: Orange (1:6) 300 2 20.2 252.5

Carrot: Orange (1:1) 400 1 35.9 444.2

Carrot: Orange (6:1) 400 3 35.9 408.1

210

3.4 Quality evaluation

3.4.1 Color characteristics

All the CIELAB coordinates L*, a* and b* were measured before and after processing and then

analyzed to determine the Chroma and the Hue angle. Results are presented in Table 26. The

statistical analyses performed on the collected data show that the process doesn’t have any impact

on the lightness (L*), the chroma or the hue angle. The same trend was observed by Ferrari et al.

(2010), who studied the effect of HPP on pomegranate juice. They found no effect of HPP

performed at 400 MPa, 500 MPa and 600 MPa at 25°C for 5 min on the lightness furthermore, the

color difference obtained after each treatment was very low. This first indicates that HPP doesn’t

affect the overall color of each juice and secondly consumer could barely see a difference in the

color of an untreated sample and a treated one. The same observation was made by Bull et al.

(2010) who found no significant effect of HPP treatment carried out at 600 MPa for 1 min on the

color difference of Navel or Valencia orange. The main pigment responsible for the color of these

juices is carotenoid, this also entails that retention of color means retention of carotenoid.

211

Table 26. Color of juice before and after processing.

pH Sample L* Chroma Hue angle

(radius) ΔE

4 Control 38.98±1.11a 75.95±1.18a 1.05±0.02a

0.96±0.52 P1 38.91±0.55a 75.74±0.35a 1.05±0.02a

5 Control 25.16±1.52b 59.78±2.43b 0.81±0.02b

1.82±0.73 P2 24.28±1.23b 58.33±2.03b 0.80±0.02b

6 Control 10.81±0.65c 37.32±1.79c 0.52±0.01c

4.43±0.74 P3 12.99±0.34c 40.64±1.12c 0.57±0.00c

(Note : P1 = 300 MPa, 2 min ; P2 = 400 MPa, 1 min ; P3 = 400 MPa, 3 min)


There was no significant difference (p>0.05) in ascorbic acid content between the untreated and

the processed sample (Table 27). These results are in line with the study conducted by Mclnerney

et al. (2007) who observed no significant impact of HPP either at 400 MPa or at 600 MPa for 2

min on carotenoids in carrots. Also, Butz et al. (2002), found no change in total carotenoid in

tomato after processing at 600 MPa at 25°C treatment for 60 min. This may be explained by the

fact that high pressure processing affects only weak bonds and not covalent bonds. As a small

molecule having a low molecular weight, carotenoids only feature covalent bonds, which may

explain why they are not affected by HPP. In addition, there is no statistical interaction between

the high pressure process and the pH in carotenoid degradation, which suggests that HPP has the

same impact on the carotenoid content regardless the pH of the product. The difference in

proportion of orange and carrot juice used to make up different juice blends with different pH was

significantly (p<0.05) different in the total carotenoid content. In fact, this study shows that an

orange-carrot blend at pH 6 contains two-fold more than the amount of carotenoids than in an

212

orange-carrot mix at pH 4 which appears consistent since carrot juice contains more carotenoids

than orange juice.

3.4.3 Ascorbic acid

Results obtained on ascorbic acid content are shown in the Table 27. As expected, these results

highlight a significant difference (p<0.05) in ascorbic acid content between samples at pH 4, 5 and

6. Since the juice blends were obtained by mixing orange and carrot juice in different proportion

to have blends with pH 4, 5 or 6, therefore sample at pH 4 contains more orange juice than sample

at pH 6. Orange juice contains more ascorbic acid than carrot juice; in the present study, higher

ascorbic acid content was found in the blend with pH 4 than in pH 6. These results show that for

each pH, there is no significant difference (p>0.05) between the control and the treated sample

which suggest that HPP doesn’t impact the ascorbic acid content of the sample. HPP has proven

to damage only weak bonds and not covalent bonds. Ascorbic acid as a small component featuring

only covalent bonds, hence it is not affected by HPP. In addition, samples were treated using mild

conditions (mild pressure for a short time, 1 to 3 minutes) which limits ascorbic acid oxidation.

These results are in line with those suggested by Bull et al. (2004). Their study on Navel and

Valencia orange juice treated by HPP at 600 MPa, 20°C for 1 min shows no significant difference

in both orange juice type between the control and the processed sample. The results below show

95%, 100% and 88% retention on ascorbic acid content at pH 4, 5 and 6 respectively. In line with

the previous results, Garcia et al. (2001), reported 99.6% of Vitamin C retention in orange (40

parts)-lemon (5 parts)-carrot (20 parts) and water (35 parts) mix after treatment at 500 MPa for 5

minutes. The difference in ascorbic acid retention in different blends might be due to the stability

of ascorbic acid at low pH than at high pH. Nevertheless, it can be noticed a slight but not

213

significant decrease in sample at pH 5 (treated at 400 MPa for 1 min) as compared to the one at

pH 6 (treated at 400 MPa for 3 min). This slight decrease may be explained by the vitamin C

oxidation by oxygen. According to the Henry’s law, when the pressure rises to 400 MPa, it

increases the amount of oxygen dissolved in juice containing water and consequently the vitamin

C oxidation. Sample at pH 6 was treated longer than the one at pH 5 which may give more time to

oxidation reactions to take place in the sample at pH 6 and consequently decrease the amount of

ascorbic acid.

Table 27. Results obtained for the quantification of ascorbic acid, carotenoid and phenolic content.

(Note: P1: 300 MPa, 2 min; P2: 400 MPa, 1 min; P3: 400 MPa, 3 min)

3.4.4 Total phenolic content

Phenolic compounds are produced by plants during secondary metabolism and are located inside

the cells. Application of high pressure creates pores in the cells, thereby allowing the

extraction/release of phenolics. In this study, it was observed that there was non-significant

(p>0.05) decrease in total phenolic compounds after high pressure processing. This observation

may be due to the mild conditions used in this study. Application of 300 or 400 MPa for 1 to 3 min

may not be a high or long enough treatment to allow a proper extraction of phenolic compounds.

pH Sample Ascorbic Acid (mg AA/100g

juice)

Carotenoid

(mg/L) Phenolic (mg/L)

4 Control 45.36±1.79a 81.39±9.60a 43.75±1.78a

P1 43.30±0.56a 65.48±11.01a 42.03±3.30a

5 Control 26.57±0.01b 163.97±41.62b 40.06±2.49a

P2 26.68±0.81b 119.27±22.12b 37.38±1.35a

6 Control 18.0±1.29c 240.79±10.20c 33.02±4.34b

P3 15.87±1.27c 224.27±33.94c 30.39±1.11b

214

The number or the size of the pores generated by HPP treatments may be too small to allow

phenolic compound to pass through the pores. These results are in line with the ones described by

Patras et al. (2009) who observed a non-significant increase in the total phenolic compound in both

strawberry and blackberry purée after HP treatment at 400 MPa for 15 min. Also, a study

performed by Landl et al. (2010) observed non-significant losses of phenolic compound in Granny

Smith apple purée at 400 MPa for 5 min at 20°C.

Besides, there was no statistical interaction between pH and the process which suggests

that there is no interference between these two parameters on the phenolic content of each sample.

This study also reveals a significant change in phenolic content between samples at pH 4-5 and

pH 6. The former ones containing more phenolic component than the later one. This may be

explained by the fact that orange juice may contain more phenolic compounds than carrot juice.

3.5 Quality changes during storage

3.5.1 Microbial growth during storage

As shown in Figure 31, initial aerobic bacterial load of unprocessed samples is about 4 log, and at

least 2 log higher than the corresponding processed sample. A statistical analysis on control sample

shows that there is a significant effect (p<0.05) of the pH on the inactivation of aerobic mesophiles

by HPP. The count obtained as initial aerobic bacteria content for the sample at pH 6 is in line with

the values quoted by Park et al. (2002); the authors stated a value of 105 to 106 CFU/mL of initial

total aerobes in raw carrot. As expected and shown by the results, the higher the pH, the higher the

aerobic bacteria load. Hence, among control sample, the one at pH 4 has the least aerobic bacteria

content. Most of aerobic bacteria have their optimum pH between 6.5 and 7.5. Therefore, high acid

215

food (pH<4.6) doesn’t favor bacteria growth compare to low acid food (pH>4.6). Besides, it can

be observed for the control samples that the aerobic bacteria load increases significantly over time.

For instance, the microbial load in blend with pH 4 (control) had one log rise in 8 days. In addition,

control samples at pH 5 and 6 were found to be spoiled after 10 days of storage at 4°C and were

discarded.

However, results obtained on processed sample are completely different. Processing

condition for blend with pH 4 was 300 MPa, 2 min and for pH 5 was 400 MPa, 1 min and lastly

for pH 6 was 400 MPa, 3 min. Total aerobic mesophiles count in all the blends after processing

was around 1 log. A statistical analysis carried out on processed sample shows that there is no

significant effect of the pH on the microbial load after processing (p>0.05) which may mean that

each selected process suits well to each product category based on their pH value. There was no

significant increase in mesophiles during the first 14 days of shelf-life, but the microbial load

increases significantly (p<0.05) in 28 days from about 10 CFU/mL to less than 100 CFU/mL, but

this is still under the maximum tolerable limit (2 log) of aerobic mesophiles in juice. Hence, high

pressure treatment allowed each blend to stay acceptable in terms of microbial load during 28

days’ storage period.

216

Figure 31. The growth of total aerobic mesophilic bacteria in the control (un-processed) and high

pressure treated juice blends during storage at 4°C

3.5.2 Changes in pH and TSS during storage

In the present study, it was observed that HPP doesn’t significantly affect pH of the product.

However, some studies show that HPP causes a decrease in pH. Since this process affects weak

bonds, it can cause dissociation of water molecules. It may also affect bonds between acids and

their associated protons leading to release protons in the solution and therefore reducing the pH.

Heremans (1995) found 0.2 units of pH drop per increase of 100 MPa in apple juice. However, in

the present study, the decrease was not significant. These results are in line with the ones found by

Bull et al. (2004), on Valencia and Navel Orange juice who didn’t find any significant decrease in

pH after treatment at 600 MPa for 1 min (initial pH of 4.28 in fresh Valencia orange juice and 4.23

after high pressure treatment). Likewise, no significant effect of high pressure treatment performed

at 600 MPa for 10 min on pH was found by Park et al. (2002) in their study on carrot juice.

0

1

2

3

4

5

6

7

0 5 10 15 20 25 30

Log

(CFU

/mL)

Days

Carrot+Orange (1:6) (Control) Carrot+Orange (1:1) (Control) Carrot:Orange (6:1) (Control)

Carrot:Orange (1:6) (HPP) Carrot:Orange (1:1) (HPP) Carrot:Orange (6:1) (HPP)

217

During storage, the pH of control sample decreases by approximately 1 unit for the blend

with pH 5 and 6 and about 0.70 unit for the blend with pH 4. Blend with pH 5 and 6 followed the

same trend: there was no significant difference in pH during the first 2 days and then there was a

significant decrease in pH between day 2 and day 4 (Figure 32). This decrease in pH could be due

to microbial growth according to Rivas et al. (2005). But there was no significant growth of

bacteria between day 2 and day 4. Therefore, acidification due to microbial growth might not be

the only cause of decrease in pH. This decrease in pH may be mostly due to chemical reactions

happening in the juice like enzymatic reactions leading to a decrease in pH. For instance, due to

enzymatic reactions in aqueous phase, the carboxylic acid function of galacturonic acid present in

the pectin can release protons to water molecules leading to the formation of H3O+ and therefore

reducing the pH. Such enzymatic reactions producing acids may explain the significant reduction

of pH in juice blends during storage.

For the processed sample, first, the pH for the sample at pH 6 remains constant during the

first week of storage whereas it remains approximately stable for 2 weeks of storage for the other

two blends (pH 4 and 5). After that, there was a significant decrease in pH until 28 days of storage.

For the sample at pH 4 there was a decrease of 13%, for pH 5 there was decrease of 18% and for

pH 6, there was 12%. The pH reduction observed may be due to activities of remaining enzymes

leading to the production of acids. The microbial growth, which starts slightly increasing more

during the third week of storage than during the first 2 weeks may also be responsible in a fair

measure of the decrease in pH.

TSS is an important parameter to take into account when processing fruit and vegetable

juice. Total soluble solid, expressed in °Bx, measures the total amount of sugar, organic acids and

218

other soluble molecules of a product. Therefore, TSS is one of the parameter which allows us to

assess the juice quality and stability when performing a shelf-life study. First of all, each process

doesn’t seem to impact the TSS which remains almost the same for the control and the processed

sample. For all control samples, it was observed that a slight but not significant (p>0.05) increase

in their total soluble solid content in 10 days. For all processed samples, it was observed that the

TSS stays constant during the 28 days of shelf-life. Therefore, the content of sugar, organic acids

and other molecules included in the TSS measurement has been quite stable in these products for

28 days. These results are in line with the ones described by Varela-Santos et al. (2011), who

reported a stable TSS value on pomegranate juice during 15 days of storage at 4°C after having

been processed at 350, 450 and 650 MPa for 30, 90 and 150 sec.

Each juice blend (pH 4, 5, and 6) had a different °Brix value and they were significantly

different (p<0.05), the higher one being for the blend with pH 4. This may be explained by the

high amount of organic acid in orange juice, besides that orange juice is comparatively sweeter

than carrot juice, hence had higher TSS.

219

Figure 32. Change in pH and TSS of the juice blends (Control and Processed) during storage at 4°C for

28 days.

3.5.3 Changes in color of juice during storage

As to the lightness, a statistical analysis shows a significant decrease only for the control sample

at pH 4 and for the treated sample at pH 6. Nevertheless, the decrease in the lightness value for the

control sample at pH 4 is very slight. Otherwise, other samples can feature a slight but not

significant decrease in their lightness. These results show that the lightness is “naturally” stable

for 10 days, although the lightness tends to decrease more in the sample at pH 4 than for the others.

The treatments performed on the sample at pH 4 and 5 seem to preserve the lightness over 28 days.

2

3

4

5

6

7

0 5 10 15 20 25 30

pH

Days

pH 4 pH 5 pH 6 pH 4 (Control) pH 5 (Control) pH 6 (Control)

6

8

10

12

14

16

0 5 10 15 20 25 30

TSS

(°B

x)

Days


220

Nevertheless, the treatment carried out on the sample at pH 6 may not allow maintaining the

lightness constant because of the significant decrease that appears during the first 14 days of

treatment.

The a* value indicates the degree of red/green color in our sample. The control samples at

pH 4 and 5 features a constant a* value over the 10 days of storage at 4°C. For the sample at pH

6, the a* value increases significantly during the storage. This sample was completely spoiled after

10 days of storage at 4°C and featured a phase separation. The aggregation of some components

such as carotenoids may have occurred in this sample leading to a higher a* value despite having

been manually homogenized before the analysis. Also, for all the treated samples, the statistical

analysis shows a significant decrease during the storage period, the samples became less and less

red. The treated sample at pH 4 undergoes a significant decrease in its a* value during the first

week of storage and then remain constant. The treated sample at pH 5 remains stable for 3 weeks

before decreasing significantly. The a* value of the sample at pH 6 decreases gradually over the

28 days of shelf-life. The decrease in the red intensity may be due to the oxidation of carotenoids

since they are the main components responsible for the red color in carrot juice. Chen et al. (1996)

reported a decrease in α- and β- carotene after 3 months of storage at 4°C in the dark. They acidified

carrot juice to pH 4 and treated them at 105°C for 30 sec. Vitamin C as an antioxidant may prevent

carotenoids from being oxidized and so, help to maintain the fruit juice color over time. The

difference in the period of storage: 10 days for the control samples and 28 days for the treated

sample may have explained why there was not any difference in the a* value of the control sample

at pH 4 and 5. Besides, carotenoid oxidation depends on many factors such as the time of storage

and the antioxidant component amount in the sample. The b* value remains constant for almost

221

all samples which meant that there was no change in the yellow/blue color of the product. Only

the treated sample at pH 6 features a decrease in b* value.

The color difference ΔE for control samples remain approximately constant over 10 days.

With respect to the treated samples, pH 4 and pH 5 samples have a constant ΔE over 28 days of

storage, which means that the color difference remains constant during this period. Bull et al.

(2004) also observed a similar increase in the color difference on Navel and Valencia orange after

treatment at 600 MPa for 1 min, and during storage at 4°C for 4 weeks. Nevertheless, treated

sample at pH 6 features a significant change in the color after 21 days of storage, which may be

explained by the significant change in its L*, a* value and b* value which might be due to the

oxidation of carotenoids which has a direct impact on the color. In fact, according to Baranska et

al. (2016), the number of conjugated bonds presents in carotenoids determines its color. The more

the molecule features double bonds, the more intense and the reddish the color. According to

Rodriguez-Amaya et al. (2015), oxidation is the main degradation process occurring during

storage. Since oxidation reduces the number of conjugated bond in carotenoids, it leads to a

decrease in color intensity. Besides, oxidation of carotenoids depends on the amount of antioxidant

compound presents in the food matrix. This may be one of the reasons why there was not any

significant difference in the ΔE of samples at pH 4 and pH 5. In fact, as a powerful antioxidant,

the ascorbic acid presents in orange juice may protect carotenoids from degradation. Also,

carotenoid is a fairly stable component which may explain why there was no any difference in

terms of color in any of the control samples. Actually, the 10 days of storage may not be long

enough to access a significant difference in their color.

222

0

5

10

15

20

25

30

35

40

45

0 5 10 15 20 25 30

L*

Days


20

25

30

35

40

45

0 5 10 15 20 25 30

a*

Days

pH 4 ph 5 pH 6 pH 4 (Control) pH 5 (Control) pH 6 (Control)

223

Figure 33. Change in color characteristic of the juice blends during storage at 4°C for 28 days.

Conclusions

The results of this study show that HPP can be used to safely process selected carrot-orange juice

blends without sacrificing quality attributes. The findings could be used as a baseline for the

commercial pasteurization of juice blends with different pH by HPP. Lower pH favors the

inactivation of microorganisms by HPP; it was found that there was less energy required to achieve

same level of inactivation for a blend with pH 4 as compared to the blend with pH 5, and 6. For

instance, 300 MPa, 2 min was enough to achieve a 5 log reduction for the blend at pH 4, whereas,

400 MPa, 1 min was required for the sample at pH 5 and 400 MPa, 3 min for the one at pH 6.

Therefore, the sample at pH 4 consumed less energy (252.5 kJ/L) than the other two samples.

Besides that, each treatment was able to retain the ascorbic acid and carotenoid content as well as

the physical characteristics (color, pH, and TSS). The storage study performed for 28 days at 4°C

after processing following the previous conditions, reveals the effectiveness of each treatment to

maintain a low level of aerobic mesophilic bacteria in each sample (less than 100 CFU/mL).

0

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30

b*

Days


224

Furthermore, during storage period, no color difference was observed for the samples at pH 4 and

5 but there was a slight change in the color for the sample at pH 6 after 21 days of storage. Hence

it can be concluded that HPP is a promising alternative technology to process juice blends with

different pH. Based on the pH of the juice, mild processing conditions for HPP can be determined;

in addition to that, energy required during processing and shelf life of the HP processed juice can

also be estimated.

225

References

Arena, E., Fallico, B., & Maccarone, E. (2001). Thermal damage in blood orange juice: kinetics

of 5‐hydroxymethyl‐2‐furancarboxaldehyde formation. International Journal of Food


Balasubramaniam, V. M., Martínez-Monteagudo, S. I., & Gupta, R. (2015). Principles and

application of high pressure–based technologies in the food industry. Annual Review of

Food Science and Technology, 6, 435-462.

Baranska, M., & Kaczor, A. (2016). Carotenoids: nutrition, analysis and technology. John Wiley

& Sons.

Bull, M. K., Zerdin, K., Howe, E., Goicoechea, D., Paramanandhan, P., Stockman, R., ... &

Stewart, C. M. (2004). The effect of high pressure processing on the microbial, physical

and chemical properties of Valencia and Navel orange juice. Innovative Food Science and


Butz, P., Edenharder, R., Garcı́a, A. F., Fister, H., Merkel, C., & Tauscher, B. (2002). Changes in

functional properties of vegetables induced by high pressure treatment. Food Research

International, 35(2), 295-300.

Chen, H. E., Peng, H. Y., & Chen, B. H. (1996). Stability of carotenoids and vitamin A during

storage of carrot juice. Food Chemistry, 57(4), 497-503.

Chen, Y., Yu, L. J., & Rupasinghe, H. P. (2013). Effect of thermal and non‐thermal pasteurisation

on the microbial inactivation and phenolic degradation in fruit juice: a mini‐

review. Journal of the Science of Food and Agriculture, 93(5), 981-986.

226

Ciancaglinia, P., Santos, H. L., Daghastanli, K.R.P., Thedei, Gerald Jr. (2001). Using a classical

method of vitamin C quantification as a tool for discussion of its role in the body.

Biochemistry and Molecular Biology Education. 29(3), 110-114

Conner, D. E., Brackett, R. E., & Beuchat, L. R. (1986). Effect of temperature, sodium chloride,

and pH on growth of Listeria monocytogenes in cabbage juice. Applied and Environmental


Derradji-Benmeziane, F., Djamai, R., & Cadot, Y. (2014). Antioxidant capacity, total phenolic,

carotenoid, and vitamin C contents of five table grape varieties from Algeria and their

correlations. OENO One, 48(2), 153-162.

Evrendilek, G. A., & Balasubramaniam, V. M. (2011). Inactivation of Listeria monocytogenes and

Listeria innocua in yogurt drink applying combination of high pressure processing and mint

essential oils. Food Control, 22(8), 1435-1441.

Fernández García, A., Butz, P., Bognàr, A., & Tauscher, B. (2001). Antioxidative capacity,

nutrient content and sensory quality of orange juice and an orange-lemon-carrot juice

product after high pressure treatment and storage in different packaging. European Food

Research and Technology, 213(4), 290-296.

Ferrari, G., Maresca, P., & Ciccarone, R. (2010). The application of high hydrostatic pressure for

the stabilization of functional foods: Pomegranate juice. Journal of Food

Engineering, 100(2), 245-253.

Heremans, K. (1995) High pressure effects on biomolecules. In High Pressure Processing of Foods

ed. Ledward, D. A., Johnston, D. E., Earnshaw, R. G. and Hasting, A. P. M. pp. 81-97.

Nottingham: Nottingham University Press

227

Kabasakalis, V., Siopidou, D., & Moshatou, E. (2000). Ascorbic acid content of commercial fruit

juices and its rate of loss upon storage. Food Chemistry, 70(3), 325-328.

Landl, A., Abadias, M., Sárraga, C., Viñas, I., & Picouet, P. A. (2010). Effect of high pressure

processing on the quality of acidified Granny Smith apple purée product. Innovative Food

Science and Emerging Technologies, 11(4), 557-564.




McInerney, J. K., Seccafien, C. A., Stewart, C. M., & Bird, A. R. (2007). Effects of high pressure

processing on antioxidant activity, and total carotenoid content and availability, in

vegetables. Innovative Food Science and Emerging Technologies, 8(4), 543-548.

Oey, I., Van der Plancken, I., Van Loey, A., & Hendrickx, M. (2008). Does high pressure

processing influence nutritional aspects of plant based food systems?. Trends in Food


Park, S. J., Lee, J. I., & Park, J. (2002). Effects of a Combined Process of High‐Pressure Carbon

Dioxide and High Hydrostatic Pressure on the Quality of Carrot Juice. Journal of Food

Science, 67(5), 1827-1834.

Patras, A., Brunton, N. P., Da Pieve, S., & Butler, F. (2009). Impact of high pressure processing




228

Polydera, A. C., Stoforos, N. G., & Taoukis, P. S. (2003). Comparative shelf life study and vitamin

C loss kinetics in pasteurised and high pressure processed reconstituted orange

juice. Journal of Food Engineering, 60(1), 21-29.

Rodriguez-Amaya, D. (2015). Food carotenoids: chemistry, biology and technology. John Wiley

& Sons.

Sahu, J. K. (2014). Introduction to advanced food process engineering. CRC Press.

Silva, F. V. (2016). Modeling the inactivation of psychrotrophic Bacillus cereus spores in beef

slurry by 600MPa HPP combined with 38–70° C: Comparing with thermal processing and

estimating the energy requirements. Food and Bioproducts Processing, 99, 179-187.

Varela-Santos, E., Ochoa-Martinez, A., Tabilo-Munizaga, G., Reyes, J. E., Pérez-Won, M.,

Briones-Labarca, V., & Morales-Castro, J. (2012). Effect of high hydrostatic pressure

(HHP) processing on physicochemical properties, bioactive compounds and shelf-life of

pomegranate juice. Innovative Food Science and Emerging Technologies, 13, 13-22.

229

CHAPTER SEVEN

PROCESSING OF CARROT-ORANGE JUICE BLEND BY PULSED ELECTRIC

FIELDS AND NISIN AT VARIOUS TEMPERATURES

Abstract

In this study, a carrot-orange juice blend (one-part carrot and six-part orange, pH 4) was processed

with pulsed electric fields under two different conditions: low electric field intensity-high

frequency (LEHF: 15 kV/cm, 979 Hz) and high electric field intensity-low frequency (HELF: 30

kV/cm, 235 Hz). The treatments combined PEF with mild heat (20, 35, and 50°C) and nisin (0,

25, and 50 ppm) to study the inactivation of L. innocua. The overall goal of this study was to

investigate and compare two different processing approaches for the inactivation of L. innocua

and total aerobic mesophilic bacteria in a carrot-orange juice blend, and to explore the resulting

changes in physicochemical characteristics of the juice after processing.

The high field-low frequency combination was found to be more efficient on the

inactivation of L. innocua. There was about a 4 log reduction of L. innocua by HELF, while LEHF

rendered less than a 3 log reduction at room temperature. When the temperature was raised to

35°C, there was about a 5 log reduction, and at 50°C there was complete inactivation (>7 log). The

combination of 50 ppm nisin and HELF at 20°C resulted in a 5 log reduction of L. innocua, while

a temperature of 35°C was required to achieve the same level of inactivation using the same

concentration of nisin and LEHF.

A non-significant increase in total carotenoids of juice was observed for both processing

approaches at all selected temperatures. There was a significant decrease (p<0.05) in ascorbic acid

230

in treated samples relative to untreated control, but no difference was observed between the

processing conditions.

The study shows that a high electric field-low frequency combination is more effective for

microbial inactivation than a low frequency-high electric field combination at the same level of

energy input. Synergism in microbial inactivation was observed when PEF treatment was

combined with nisin, heat, or both.

1. Introduction

Pulsed electric fields (PEF) is a nonthermal technology that has shown promising results in the

processing of liquid and semi-liquid food. In many cases, PEF is able to inactivate microorganisms

without significantly affecting heat-sensitive bioactive compounds. PEF processing systems are

composed of a high-power pulse generator, a treatment chamber with electrodes, a pump, and an

oscilloscope. PEF systems apply short pulses of electricity, with a discharge of electrons over only

a few microseconds. These short pulses inactivate microorganisms with only minor effects on food

quality attributes. The electric fields generate a voltage difference across the microbial cell

membrane, creating a very intense force promoting the formation of pores due to electrical

breakdown. These pores allow the passage of essential cell components associated with its

metabolism, and thus promotes the inactivation of the microorganisms. The electric pulses are also

responsible for modifying intracellular enzymes and detaching the cytoplasm from the membrane,

two additional factors which also significantly contribute to microbial inactivation.

PEF systems involve multiple variables such as electric field intensity, frequency, flow

rate, pulse width; these factors all have effects on microbial inactivation. Two commonly used

231

types of thermal process protocol are high temperature short time (HTST) and low temperature

long time (LTLT) processing. Studies have shown that although both processes are efficient in

microbial inactivation, HTST allows greater nutrient retention. Along the same lines as HTST and

LTHT, this study compares two different PEF approaches: high electric field-low frequency

(HELF, 30 kV/cm, 235 Hz) and low electric field-high frequency (LEHF, 15 kV/cm, 979 Hz).

Electric field intensity (low or high) is the strength of the field generated due to an applied voltage

between two electrodes. Pulse frequency directly correlates with the total number of pulses and

treatment time.

The objective of this study was to compare two different PEF processing approaches

(HELF and LEHF) in combination with mild temperatures (20, 35, and 50°C), and nisin at two

concentrations (25 and 50 ppm). The comparisons were made on the basis of Listeria innocua

inactivation, as well as retention of physicochemical characteristics and bioactive compounds.


2.1 Sample preparation

Commercially available pre-pasteurized carrot juice (Bolthouse Farms, Bakersfield, CA) and

orange juice (Simply Orange Juice Company, Apopka, FL) were purchased from a local

supermarket. The pre-pasteurized juice was used for microbiological testing primarily to avoid

interactions between inoculated microorganisms and natural microflora. Nisin-A (Handary S.A,

Brussels, Belgium) was added to the juice in the concentrations of 0, 25, and 50 ppm to study

possible synergism between PEF and nisin, as well as between PEF, nisin, and temperature. For

subsequent testing to evaluate quality, fresh carrot-orange juice was prepared in the lab using a

232

domestic juice extractor where the ingredients were purchased from a local supermarket. Fresh

juice was used in this phase to evaluate the effect of the selected process on the quality attributes

where the fresh juice blend had a pH of 4.02 and TSS of 11.10°Brix.

2.2 Culture preparation and inoculation

A freeze-dried culture of Listeria innocua (ATCC 8456TM) was purchased from American Type

Culture Collection. The dried culture was grown in tryptic soy broth with 0.6% yeast extract

(TSBYE) (Becton Dickinson, Sparks, MD, USA) at 37°C, agitating at 225 rpm in a water bath

shaker. Absorbance was measured every hour at 560 nm. Cells were harvested in the early

stationary phase, mixed with sterile 20% glycerol, and stored at -18°C. During experiments, frozen

culture was activated in TSBYE and inoculated in the juice so as to have an initial load of around

7 log.

2.3 PEF processing

A PEF system designed by Diversified Technologies Inc. (DTI, Bedford, MA, USA) was used in

this study. Two different processing conditions were selected to investigate and compare the effect

of LEHF and HELF on the inactivation of microorganisms, analogous to two conventional thermal

pasteurization methods, HTST and LTHT. The selection of processing conditions (Table 28) was

based on the electrical conductivity of the product (4.2 mS/cm) and arcing by the system during

processing. Both conditions had a power input of 1 kW, and the product inlet temperature during

processing was set to 20, 35, and 50°C. For LEHF, the electric field intensity was 15 kV/cm, and

the frequency was 979 Hz, corresponding to 75 pulses; for HELF, the electric field intensity was

30 kV/cm, and the frequency was 235 Hz, corresponding to 18 pulses. The flow rate was 400

233

mL/min, with a pulse width of 2 µs in both processes. All treatments were conducted at three

different product inlet temperatures: 20, 35, and 50°C, and the inlet temperature of the juice was

adjusted using a heat exchanger (Microtherm temperature control system Chromalox CMX-250

12C, Chromalox, Inc., Edinboro, PA, USA) There was around a 10 and 11°C rise in temperature

after LEHF and HELF, respectively.

Table 28. Processing parameters for PEF

Processing parameters

Flow rate (mL/min) 400

Electric Field Intensity (kV/cm) 15 and 30

Voltage (kV) 9.8 and 19.5

Treatment time (μs) 36 and 150

Frequency (Hz) 235 and 979

Pulse width (μs) 2

Temperatures (°C) 20, 35, and 50

Nisin concentration (ppm) 0, 25, and 50

2.4 Analysis of survivors

After processing, treated juice was collected in sterile sealable pouches. One mL of the sample

was serially diluted in 0.1% sterile peptone water. Pour plating was done in tryptic soy agar with

234

0.6% yeast extract (Difco, Becton, Dickinson and Co., Sparks, MD). The plates were incubated

for 24 hours at 37 °C, and the colonies were counted manually and expressed as CFU/mL.


2.5.1 Total aerobic mesophiles

To quantify total aerobic mesophilic bacteria in fresh and processed juice, serial dilutions of the

juice were made with 0.1% peptone water. Samples were pour-plated using Plate Count Agar

(Difco, Becton, Dickinson and Co., Sparks, MD) and incubated at 37°C for 48 hours; mesophiles

were counted manually and expressed as log (CFU/mL).

2.5.2 pH, TSS, and color

The pH of the juices was measured directly using a pH meter (Orion 4-star, Thermo Fisher

Scientific, Waltham, MA, USA) at 21ºC. Color parameters (Lightness, L*; redness, a*; and

yellowness, b*) of the juices were measured in a cuvette using a colorimeter (Konica Minolta,

CM-5, New Jersey, USA). The total soluble solid (TSS) content of the juice was determined using

a hand-held refractometer (Atago No. 480, Atago Inc. Tokyo, Japan) at 21°C.


The total carotenoid content of juices was analyzed using the method described by Liao et al.

(2007). In brief, 25 mL of juice was mixed with 80 mL of a hexane/acetone mixture (1:1, v/v) in

a separating funnel. The organic phase was separated from the aqueous phase and collected and

its absorbance was measured at 450 nm using a spectrophotometer (Spectronic 20 Genesys,

Spectronic Instruments Inc., Rochester, NY, USA). The total carotenoid content was determined

235

using the standard curve of β-carotene, and the results were expressed as mg β-carotene per liter

of sample.

2.5.4 Ascorbic acid

The amount of ascorbic acid in fresh and processed juices was determined by a redox titration

method using iodine and starch indicator according to the method described by Ciancaglini et al.

(2001) with some modifications. A juice sample (20 mL) was pipetted into a 250-mL conical flask

and 150 mL of distilled water and 1 mL of starch indicator were added to the flask. The sample

solution was titrated with a 0.005 mol/L iodine solution until formation of the first permanent

green-black color was observed.

2.6 Energy calculations

The energy input for each process was determined using the fundamental relation between power,

voltage, and current (Eq. 1)

𝑃 = 𝑉. 𝐼. 𝑥

𝑡 (1)

Where P is energy input (kW), V is the input voltage (kV), I is the current intensity (amp), x is the

pulse width (μs), and t is the treatment time (μs).

2.7 Data analysis

All data were recorded and analyzed using Microsoft Excel 2016. Analysis of variance (ANOVA)

between different processes was studied using Minitab (Minitab Inc. State College, PA). A p-value

of 0.05 was chosen as the limit for significance.

236

3. Results and discussion

3.1 Rise in temperature during processing

There was a rise of about 10°C in LEHF, and about 11°C increase for HELF (Table 29). The

increase in temperature of the product during PEF processing is mainly due to the Joule effect

(Buckow et al., 2013). This increase in temperature can have advantages, as it can act together

with PEF to facilitate inactivation of microorganisms, but sometimes it is undesirable, as in the

case of heat-sensitive products. For processing heat-sensitive products, the temperature can be

controlled using a heat exchanger between two sets of electrodes, with immediate cooling of the

product after processing. The rise in temperature in PEF processing depends on the electrical

conductivity of the product being processed; in general, higher electrical conductivity leads to a

greater rise in temperature. As the electrical conductivity of the product also depends on

temperature, its electrical conductivity increases during processing as the temperature rises, and in

turn the current increases. This is not always desirable, as the system does not work when electric

current exceeds a limit. Hence, the proper balance between temperature rise and other treatment

factors needs to be considered.

Table 29. Temperature rise during pulsed electric field processing of carrot-orange juice blends

Process Initial temperature (°C) Final temperature (°C) ΔT (°C)

PI+20°C 20.4±1.7 30.7±3.5 10.2

PII+20°C 20.8±1.4 32.1±1.3 11.3

PI+35°C 35.2±0.4 45.5±0.5 10.3

237

PII+35°C 35.0±0.1 46.4±0.4 11.3

PI+50°C 50.2±0.6 61.1±1.1 10.9

PII+50°C 50.6±1.1 62.2±0.7 11.6

(PI: LEHF; PII: HELF)

3.2 Combined effect of PEF and heat

The application of LEHF or HELF significantly reduced (p<0.05) the count of L. innocua in the

carrot-orange juice blend. At room temperature (20°C), there was around 3 log reduction after

treatment with LEHF and approximately 4 log reduction after treatment with HELF combinations

(Figure 34). The high field-low frequency combination was found to be slightly more efficient for

inactivation of L. innocua. Results from the present study are in line with previous findings;

Mosqueda-Melgar et al. (2007) observed 3 log reductions of L. innocua at neutral pH using electric

field intensities between 30 and 50 kV/cm and 100 pulses. The present study was carried out with

a lower number of pulses than that study, but due to the acidity of carrot-orange juice (pH 4), the

level of inactivation was equivalent.

From the present study, it can be understood that electric field intensity plays a greater role

than pulse frequency in microbial inactivation. Iu et al. (2001) observed that when electric field

intensity was increased from 60 to 80 kV/cm, there was an improvement in microbial reduction

from 0.1 to 1 log, but changing the number of pulses had no significant effect on microbial

reduction. In contrast, Aronsson et al. (2001) found that not only EFI but also pulse duration and

the number of pulses had significant effects on the level of inactivation of gram-positive and gram-

negative bacteria. It is worth noting that the number of pulses is directly correlated with frequency,

238

i.e. as frequency increases, more pulses will be applied; hence, in PEF processing more weight has

to be given to electric field intensity (EFI) than to any other variable. In other words, EFI must be

used at the maximum possible level that the system allows, while keeping frequency, treatment

time, and number of pulses at an optimum level.

Temperature has been shown to have a critical effect on PEF processing with and without

additional hurdles such as antimicrobials (Heinz et al., 2003; Sepulveda et al., 2005; Siemer et al.,

2014). There was a significant difference (p<0.05) in the survivor fraction between the three

temperatures, and between the two different processing approaches. A greater reduction in counts

was observed when the temperature of juice was increased from 20 to 35°C. HELF provided a

3.96 log reduction at 20°C; when the temperature was raised to 35°C there was a 4.69 log

reduction, and at 50°C complete inactivation was observed (>7 log, below the limit of detection).

In the case of LEHF (15 kV/cm and 979 Hz), there was a 2.96 log reduction at ambient

temperature, and a 4.15 log reduction at 35ºC. From these results, it can be concluded that

incorporation of mild heat in PEF processing greatly enhance the efficacy of PEF in microbial

inactivation.

The application of heat can cause significant changes in the membrane properties of

microbial cells. Heat may cause phase transitions of phospholipids present in the membrane,

changing normally rigid cells to a liquid-crystalline phase (Stanley, 1991; Koga, 2012). This

change in phase, combined with reductions in the thickness of cell membrane due to charge

attraction across the membrane, can work together to cause permeabilization of the cell at lower

electric field intensities when PEF is conducted at elevated temperatures (Jayaram et al., 1993).

239

Hence, at higher temperatures (>20°C) target log reduction can be achieved with lower electric

fields or less specific energy input, which may improve the efficacy of the process.

Figure 34. Inactivation of L. innocua and naturally occurring mesophiles in carrot-orange juice blends by

pulsed electric fields. (ND: Not Detectable, below the detection limit) (PI: LEHF; PII: HELF)

3.3 Combined effects of PEF and nisin

A greater reduction in Listeria innocua counts was observed when PEF (either LEHF or HELF)

was combined with nisin (25 or 50 ppm) as compared to apply only PEF. There was a synergistic

interaction between PEF treatments and nisin on the inactivation of L. innocua; PEF alone caused

<4 log reductions, whereas PEF (HELF) and nisin (25 ppm) combined caused a greater than 4 log

reduction.

The physical changes nisin causes in the cell membrane of L. innocua may have increased

its sensitivity to PEF treatment. Nisin is an antimicrobial peptide that can form ion-permeable

pores in cell membranes (Smith et al., 2002). The effect of incorporation of 25 ppm nisin, as well

as increasing the nisin concentration from 25 ppm to 500 ppm, were both significant (p<0.05) in

the inactivation of L. innocua. At room temperature, the incorporation of nisin had a more

0

2

4

6

8

10

Control PI+20°C PII+20°C PI+35°C PII+35°C PI+50°C PII+50°C

L. in

no

cua

(Lo

g C

FU

/mL

)

ND ND

240

pronounced effect on microbial inactivation when combined with a HELF process. The target 5

log reduction was achieved by using 50 ppm nisin at 20°C under HELF conditions. Results from

present study in line with the finding of Hodgins et al. (2002) and Liang et al. (2002). Those authors

found synergy in combining PEF and nisin for microbial inactivation in orange juice. In a study

on milk processing by PEF, synergism on the inactivation of L. monocytogenes was found between

PEF and nisin at a concentration of 25 or 50 IU/mL (Maisnier-Patin et al., 1995). Synergy between

PEF and nisin was also observed by Iu et al. (2001), who found a significantly greater reduction

of E. coli in apple juice when nisin was added before PEF treatment as compared to apply only

PEF. This synergy between PEF and nisin might be due to sub-lethal injury caused by PEF and

thus increasing the sensitivity of microbial cells to nisin, and enhancing the inactivation (Hauben

et al., 1996).

3.4 Combined effect of PEF, heat, and nisin

In the present study, the temperature required to achieve a 5 log reduction of L. innocua was

decreased from 50 to 35°C by the addition of 50 ppm nisin under LEHF conditions (Table 30). In

the case of the HELF process, a 5 log reduction was achieved at room temperature (20°C) by the

incorporation of 50 ppm nisin; for the same process at 35°C, 25 ppm of nisin was sufficient to

achieve the same level of inactivation. This clearly indicates that the efficiency of PEF is enhanced

when nisin and mild heat are incorporated. The phase transition of the microbial cell membrane

due to applied heat might have facilitated PEF in creating pores. This phase transition might have

helped nisin to reach its site of action, where it can form more pores in the cytoplasmic membrane

hastening cell death.

241

Table 30. Combined effect of PEF, mild heat, and nisin on the inactivation of L. innocua in a

carrot-orange juice blend

Processes Nisin (ppm) Log Reduction

PI+20°C

0 2.96

25 3.48

50 4.10

PII+20°C

0 3.96

25 4.21

50 5.04

PI+35°C

0 4.15

25 4.22

50 5.16

PII+35°C

0 4.69

25 5.39

50 7.50

PI+50°C

0 7.50

25 7.50

50 7.50

242

PII+50°C

0 7.50

25 7.50

50 7.50

(Note: PI: LEHF; PII: HELF)


Two different processing approaches at different temperatures were compared to demonstrate their

effects on the inactivation of naturally occurring mesophilic bacteria and some selected

physicochemical quality attributes.

3.5.1 Effect of processes on aerobic mesophiles

Before processing, there was about a 4 log of mesophiles in fresh carrot-orange juice blend. At

room temperature, HELF was slightly better than LEHF for their inactivation. There was a

significant difference between the three temperatures in mesophilic inactivation. Application of

HELF at 35°C was enough to reduce the mesophilic load to a safe level (<2 log).

243

Figure 35. Inactivation of naturally occurring mesophilic bacteria in carrot-orange juice by PEF

and mild heat. (PI: LEHF; PII: HELF)

3.5.2 Effect on pH, TSS, and Color

There was a slight decrease in the pH value of juice after PEF processing, but a significant

reduction (p<0.05) was only observed after treatment with HELF at 50°C. The reduction in pH

must be due to the electroporation of the plant tissues by PEF (Knorr et al., 2001), resulting in the

extraction of intracellular constituents, for example, organic acids. Significant (p<0.05) reductions

in TSS were observed in all treated samples relative to control. Reduction of TSS is also likely due

to tissue electroporation.

There was an increase in lightness value during PEF processing under all treatment

conditions. A significant increase (p<0.05) in L* was only observed in the case of HELF treatment

combined with heat (35°C or 50°C); with other combinations, the changes were non-significant

(p>0.05). Chroma values also increased after treatment, and a significant increase (p<0.05) was

observed at 50°C for both processes. At 35°C, a significant increase (p<0.05) was found only under

0

1

2

3

4

5

Control PI+20°C PII+20°C PI+35°C PII+35°C PI+50°C PII+50°C

Mes

op

hil

es (

Lo

g C

FU

/mL

)

244

HELF conditions. There was also an increase in hue angle under all treatment conditions, but a

significant increase was only observed for HELF at 35 and 50°C.

Table 31. Change in pH and TSS of juice before and after PEF processing

Sample pH TSS (°Bx)

Control 4.02±0.01a 11.10±0.44a

PI+20°C 3.98±0.06ab 9.80±0.50b

PII+20°C 3.95±0.07ab 9.88±0.29b

PI+35°C 3.97±0.04ab 9.58±0.39b

PII+35°C 3.94±0.02ab 9.00±0.35b

PI+50°C 3.94±0.04ab 9.85±0.30b

PII+50°C 3.89±0.04b 9.35±0.64b

PI: Low Electric Field High Frequency, LEHF; PII: High Electric Field-Low Frequency, HELF;

different superscript letters after standard deviation in the same column indicates data are

significantly different (p<0.05)

The increase in Lightness, Chroma, and hue value indicates extraction of intracellular

constituents of juice during processing. When both processing approaches are compared for each

temperature, it can be observed that HELF caused greater total color changes than LEHF. Hence,

it can be said that HELF has created greater degree of change in color as compared to LEHF at all

245

processing temperatures. When treatment at three different temperatures is compared, the change

in color value can be seen to increase with increased temperature. The change in color can have

different meanings: degradation of heat sensitive compounds, browning, enzymatic reaction, or

extraction of intracellular constituents from the minute cells of carrot present in the juice. As the

change in color may indicate degradation of nutrients, further analysis is required to identify if the

process has caused any changes in the major bioactive compounds; those results are discussed in

the following section.

Table 32. Change in color characteristics of juice before and after PEF treatment

Sample Lightness (L*) Chroma (C*) Hue (h*) ΔE

Control 30.69±0.94c 64.71±1.22b 0.95±0.02c -

PI+20°C 31.88±0.70bc 66.25±0.85ab 0.97±0.01bc 2.46±2.55b

PII+20°C 32.29±0.58abc 66.66±0.69ab 0.98±0.01abc 3.22±1.04ab

PI+35°C 32.46±0.87abc 66.94±1.03ab 0.98±0.01abc 3.51±1.16ab

PII+35°C 34.24±1.53a 68.55±1.55a 1.01±0.02a 6.71±2.71a

PI+50°C 32.74±0.96abc 67.32±1.03a 0.98±0.02abc 4.02±1.14ab

PII+50°C 33.82±1.01ab 68.35±0.97a 1.00±0.02ab 5.98±1.49ab

(Different superscript letters after standard deviation in the same column indicates data are

significantly different (p<0.05))

246

3.5.3 Effect on total carotenoid content and ascorbic acid

There was a slight increase in the carotenoid content of juice after PEF treatments. The HELF

process extracted more carotenoids than LEHF; nevertheless, both treatments did not degrade

significant amounts of carotenoid compounds regardless of processing temperature. There was a

significant reduction (p<0.05) in the ascorbic acid contents of all processed samples compared to

the control, but no significant difference (p>0.05) was observed between two different methods

and three different temperatures. The retention of ascorbic acid was about 92%. Hodgins et al.

(2002), found similar retention of ascorbic acid (97.5%) in orange juice when using PEF at 80

kV/cm, 20 pulses. Sharma et al. (1998), also found 96% retention of ascorbic acid in orange juice

treated by PEF (28 kV/cm, treatment time 100 µs). It can be concluded that combination of PEF

with heat treatment could preserve the quality attributes of the juice.

Figure 36. Effect of selected process on carotenoid and ascorbic acid content of juice

4. Conclusions

The study shows a high field-low frequency combination is superior to low frequency-high field

combination for microbial inactivation by PEF, although the energy input was equivalent. When

0

100

200

300

400

500

Car

ote

no

id (

mg/L

)

0

10

20

30

40

Asc

orb

ic A

cid

(m

g/1

00

g)

247

developing processing approaches for any juice, the focus should be on identifying the proper

electric field intensity while keeping frequency/number of pulses at an optimal level. Although

combinations of PEF and heat were able to reduce the microbial load by 5 log, the addition of nisin

reduced the required processing temperature, improving the efficiency of the process. For example,

using nisin, the required level of inactivation was achieved at ambient temperatures, while without

nisin, the target inactivation level was achieved at 50°C. Results from present study shows that the

suitable combinations of PEF, nisin, and heat can meet the FDA’s requirement of a 5 log reduction.

A combination of PEF, nisin, and heat could be used on an industrial scale for pasteurization of

other juices with similar pH and electrical conductivity; this will not only preserve juice quality,

but also make the process more energy efficient and capable of higher throughput.

248

References

Aronsson, K., Lindgren, M., Johansson, B. R., & Rönner, U. (2001) Inactivation of

Microorganisms Using Pulsed Electric Fields: The Influence of Process Parameters on

Escherichia Coli, Listeria Innocua, Leuconostoc Mesenteroides and Saccharomyces

Cerevisiae. Innovative Food Science and Emerging Technologies 2 (March): 41–54

Buckow, R., Ng, S., & Toepfl, S. (2013). Pulsed electric field processing of orange juice: a review

on microbial, enzymatic, nutritional, and sensory quality and stability. Comprehensive

Reviews in Food Science and Food Safety, 12(5), 455-467.

Hauben, K. J., Wuytack, E. Y., Soontjens, C. C., & Michiels, C. W. (1996). High-pressure transient

sensitization of Escherichia coli to lysozyme and nisin by disruption of outer-membrane

permeability. Journal of Food Protection, 59(4), 350-355.

Heinz, V, Toepfl, S. & Knorr, D. (2003) Impact of Temperature on Lethality and Energy

Efficiency of Apple Juice Pasteurization by Pulsed Electric Fields Treatment. Innovative

Food Science and Emerging Technologies 4 (June): 167–75.

Hodgins, A. M., Mittal, G. S., & Griffiths, M. W. (2002). Pasteurization of Fresh Orange Juice

Using Low‐Energy Pulsed Electrical Field. Journal of Food Science, 67(6), 2294-2299.

Iu, J., Mittal, G. S., & Griffiths, M. W. (2001). Reduction in levels of Escherichia coli O157: H7

in apple cider by pulsed electric fields. Journal of Food Protection, 64(7), 964-969.

Jayaram, S., Castle, G. S. P., & Margaritis, A. (1993). The effects of high field DC pulse and liquid

medium conductivity on survivability of Lactobacillus brevis. Applied Microbiology and

Biotechnology, 40(1), 117-122.

249

Knorr, D., Angersbach, A., Eshtiaghi, M. N., Heinz, V., & Lee, D. U. (2001) Processing Concepts

Based on High Intensity Electric Field Pulses. Trends in Food Science and Technology 12

(3): 129–35.

Koga, Y. (2012). Thermal adaptation of the archaeal and bacterial lipid

membranes. Archaea, 2012.

Liang, Z., Mittal, G. S., & Griffiths, M. W. (2002). Inactivation of Salmonella typhimurium in

orange juice containing antimicrobial agents by pulsed electric field. Journal of Food

Protection, 65(7), 1081-1087.

Maisnier-Patin, S., Tatini, S. R., & Richard, J. (1995). Combined effect of nisin and moderate heat

on destruction of Listeria monocytogenes in milk. Le Lait, 75(1), 81-91.

Mosqueda-Melgar, J., Raybaudi-Massilia, R. M., & Martín-Belloso, O. (2007). Influence of

treatment time and pulse frequency on Salmonella enteritidis, Escherichia coli and Listeria

monocytogenes populations inoculated in melon and watermelon juices treated by pulsed

electric fields. International Journal of Food Microbiology, 117(2), 192-200.

Sepulveda, D. R., Góngora-Nieto, M. M., San-Martin, M. F. & Barbosa-Cánovas, G. V. (2005)

Influence of Treatment Temperature on the Inactivation of Listeria Innocua by Pulsed

Electric Fields. LWT - Food Science and Technology 38 (2): 167–72.

Sharma, S. K., Zhang, Q. H., & Chism, G. W. (1998). Development of a protein fortified fruit

beverage and its quality when processed with pulsed electric field treatment. Journal of

Food Quality, 21(6), 459-473.

250

Siemer, Claudia, Stefan Toepfl, & Volker Heinz. (2014) Inactivation of Bacillus Subtilis Spores

by Pulsed Electric Fields (PEF) in Combination with Thermal Energy II. Modeling

Thermal Inactivation of B. Subtilis Spores during PEF Processing in Combination with

Thermal Energy. Food Control 39 (Supplement C): 244–50.

Smith, K., Mittal, G. S., & Griffiths, M. W. (2002). Pasteurization of milk using pulsed electrical

field and antimicrobials. Journal of Food Science, 67(6), 2304-2308.

Stanley, D. W., & Parkin, K. L. (1991). Biological membrane deterioration and associated quality

losses in food tissues. Critical Reviews in Food Science and Nutrition, 30(5), 487-553.

251

FINAL REMARKS

The effective combination of ultrasound and mild temperature could be used to pasteurize

low-acid juices in a shorter time as compared to thermal processing and without

significantly affecting the physicochemical characteristics

Mathematical modeling of the survivor’s curve shows both Weibull and a biphasic model

to be good fits to predict the inactivation of E. coli by ultrasound and thermal energy

The simultaneous application of ultrasound, mild temperatures, and nisin for the processing

of L. innocua inoculated carrot juice was found to be very effective as compared to apply

only ultrasound

The use of HPP in conjunction with nisin and mild temperature elevation is an effective

way of reducing microbial load in carrot juice, and potentially in other low-acid juices

The combined effect of high pressure, nisin, and mild temperature was better in microbial

inactivation than to apply only high pressure. This type of combined treatment can greatly

reduce energy consumption during processing.

HPP could be industrially used to safely process selected carrot-orange juice blends

without sacrificing quality attributes. Addition of orange juice to carrot juice results on less

processing time, reduced processing pressure and thus, lowering processing costs and

energy input. This study could also act as a baseline for the commercial pasteurization of

juice blends by high pressure at different pHs.

Pulsed electric fields processing of carrot-orange juice blends shows that high electric

field-low frequency combinations were superior to the low frequency-high field ones in

252

microbial inactivation although the power input was the same. Results also show higher

extraction of intracellular components by the application of HELF than LEHF

combinations.

For the same power input, pulsed electric fields intensity shows higher efficacy than pulse

frequency where the latter is related to treatment time and number of pulses. At the time of

developing a method for the treatment for any juice, the focus should be in identifying the

proper electric field intensity while keeping frequency/number of pulses at an optimum

level.

253

FUTURE STUDIES

The following area need further research to successfully use the combined technologies at the

commercial level:

In depth study on the application of HPP, PEF, and US together with natural antimicrobials

to inactivate pathogens.

Evaluation of sensory properties of carrot juice and blends treated with HPP, PEF, and US

and comparison with the thermally processed one.

Conduct in-depth studies on the shelf life prediction of carrot juice treated by those

technologies together with other hurdles.

Study on the effect of pulp and particulates on the inactivation of microorganisms by

selected nonthermal technologies together with hurdles.

processing carrot juice by selected nonthermal technologies

Documents

Transcript of processing carrot juice by selected nonthermal technologies